FEBS Letters 589 (2015) 1376–1382
journal homepage: www.FEBSLetters.org
EGFR/ERBB receptors differentially modulate sebaceous lipogenesis Maik Dahlhoff a, Emanuela Camera b, Matteo Ludovici b, Mauro Picardo b, Udo Müller c, Heinrich Leonhardt c, Christos C. Zouboulis d, Marlon R. Schneider a,⇑ a
Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy c Human Biology and BioImaging, Department of Biology II, LMU Munich, Germany d Departments of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center, Dessau, Germany b
a r t i c l e
i n f o
Article history: Received 5 December 2014 Revised 12 February 2015 Accepted 4 April 2015 Available online 15 April 2015 Edited by Laszlo Nagy Keywords: Sebaceous gland Epidermal growth factor receptor ERBB Sebaceous lipogenesis
a b s t r a c t The roles of the epidermal growth factor receptor (EGFR) in sebaceous glands remain poorly explored. We show that human sebocytes express EGFR and lower levels of ERBB2 and ERBB3, all receptors being downregulated after the induction of lipid synthesis. Nile red staining showed that siRNA-mediated downregulation of EGFR or ERBB3 increases lipid accumulation, whereas ERBB2 downregulation has no effect. Spectrometry confirmed induction of triglycerides after EGFR or ERBB3 downregulation and revealed induction of cholesteryl esters after downregulation of EGFR, ERBB2 or ERBB3. Thus, EGFR/ERBB receptors differentially modulate sebaceous lipogenesis, a key feature of sebaceous gland physiology and of several skin diseases. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Sebaceous glands (SG) are exocrine glands of the skin most commonly found in association with a hair follicle. During their differentiation, sebocytes progressively synthesize and accumulate lipids within their cytoplasm, and are eventually dislodged from the periphery to the core of the gland. Once a certain size is attained, the cell disrupts and releases its contents, a process termed holocrine secretion. The synthesized lipids and cellular debris reach the skin surface via the hair follicle canal, and represent the major component of skin sebum [1–3]. Although several functions have been credited to sebum, including a role in epidermal barrier and in hair follicle integrity, as well as antibacterial and antioxidant properties [4,5], its exact role remains unclear. However, increased sebum production is a key component the most common skin disease, acne [6,7]. Current data indicate that not only the amount of sebum but also the sebum composition plays a major Author’s contributions: Conception and design: M. Dahlhoff, E. Camera, M.R. Schneider. Acquisition of data: M. Dahlhoff, E. Camera, M. Ludovici, U. Müller, M.R. Schneider. Contribution of essential reagents: M. Picardo, H. Leonhardt, C.C. Zouboulis. Analysis and interpretation of data: M. Dahlhoff, E. Camera, M.R. Schneider. Writing, review, and/or revision of the manuscript: M.R. Schneider. ⇑ Corresponding author at: Gene Center, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany. E-mail address: [email protected] (M.R. Schneider).
role in the induction of sebaceous gland-associated skin diseases [8]. Sebocyte differentiation and sebaceous lipogenesis are controlled by numerous hormones, growth factors, and transcription factors. There is growing evidence suggesting that the epidermal growth factor receptor (EGFR) and its ligands play an important role in this process. The EGFR is strongly expressed in the undifferentiated sebocytes at the periphery of mouse [9] and human [10] sebaceous glands. Stimulation of hamster sebocytes with EGFR ligands increased proliferation and inhibited sebaceous lipogenesis [11], and the ligand EGF also impaired lipogenesis in human sebaceous glands maintained in vitro [12]. Interestingly, although EGF is a common medium additive for the culture of isolated human sebocytes, it does not influence their proliferation [13], which points to species-specific differences in the response of sebocytes to EGF. More recently, enlarged SGs and increased sebum production were reported in transgenic mice overexpressing the EGFR ligand epigen [14] and in Dsk5 animals, a mouse line in which the EGFR is constitutively activated independently of ligand binding [15]. The EGFR (also known as ERBB1 or HER1) belongs to a family of tyrosine kinase receptors that also includes ERBB2 (NEU, HER2), ERBB3 (HER3), and ERBB4 (HER4) [16,17]. In contrast to the EGFR, the expression and function of these related receptors in sebocytes has not been studied in detail before. Notably, a marked
http://dx.doi.org/10.1016/j.febslet.2015.04.003 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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enlargement of sebaceous glands was also reported in mice overexpressing ERBB2 [9], suggesting that ERBBs may be able to modulate sebocyte differentiation as well. Thus, the aim of this work is to study the expression and function of the ERBB receptors in sebocytes to uncover their potential functions in the biology of sebaceous glands.
Table 1 Primers and probes employed for the quantitative RT-PCR analysis. EGFR
Forward primer Reverse primer Universal probe
50 -gccttgactgaggacagca-30 50 -tttgggaacggactggttta-30 #69 (cat. No. 04688686001)
ERBB2
Forward primer Reverse primer Universal probe
50 -tgctgtcctgttcaccactc-30 50 -tcatcctcatcatcttcacattg-30 #67 (cat. No. 04688660001)
ERBB3
Forward primer Reverse primer Universal probe
50 -ctgatcaccggcctcaat-30 50 -ggaagacattgagcttctctgg-30 #37 (cat. No. 04687957001)
SZ95 sebocytes [18] were routinely cultured in SebomedÒ medium (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (Biochrom) and 5 lg/l EGF (Biochrom). For the experimental induction of lipogenesis, cells were cultured in the medium above lacking EGF for 24 h before supplementation with linoleic acid (LA; 104 M, Merck, Darmstadt, Germany) or vehicle only (DMSO) for the indicated periods of time.
ERBB4
Forward primer Reverse primer Universal probe
50 -gagcaagaattgactcgaatagg-30 50 -ttcctgacatgggggtgtag-30 #63 (cat. no. 04688627001)
TGFA
Forward primer Reverse primer Universal probe
50 -ttgctgccactcagaaacag-30 50 -atctgccacagtccacctg-30 #63 (cat. No. 04688627001)
AREG
Forward primer Reverse primer Universal probe
50 -cggagaatgcaaatatatagagcac-30 50 -caccgaaatattcttgctgaca-30 #38 (cat. No. 04687965001)
2.2. siRNA-mediated downregulation
EREG
Forward primer Reverse primer Universal probe
50 -tggtctcttcactcaggtctca-30 50 -cgtgagttggcatagggaac-30 #86 (cat. No. 04689119001)
HBEGF
Forward primer Reverse primer Universal probe
50 -tggggcttctcatgtttagg-30 50 -catgcccaacttcactttctc-30 #55 (cat. No. 04688520001)
EGF
Forward primer Reverse primer Universal probe
50 -aagaatgggggtcaaccagt-30 50 -tgaagttggttgcattgacc-30 #27 (cat. No. 04687582001)
BTC
Forward primer Reverse primer Universal probe
50 -actgcatcaaagggagatgc-30 50 -tctcacaccttgctccaatg-30 #49 (cat. No. 04688104001)
EPGN
Forward primer Reverse primer Universal probe
50 -tttgggagttccaatatcagc-30 50 -tgtgattggaggtgttacagtca-30 #34 (cat. No. 04687671001)
SREBF1
Forward primer Reverse primer Universal probe
5’-cgctcctccatcaatgaca-‘3 5’-tgcgcaagacagcagattta-‘3 #77 (cat. no. 04689003001)
FAS
Forward primer Reverse primer Universal probe
5’-gtggacccgctcagtacg-‘3 5’-ggacgataatctagcaacagacg-‘3 #60 (cat. no. 04688589001)
DGAT2
Forward primer Reverse primer Universal probe
5’-caagcccatcaccactgtt-30 50 -tcgatgtcttgctgggttg-30 #78 (cat. No. 04689011001)
FADS2
Forward primer Reverse primer Universal probe
50 -ctacgctggagaagatgcaa-30 50 -ttcaagaacttgcccacga-30 #85 (cat. No. 04689097001)
SCD1
Forward primer Reverse primer Universal probe
50 -cctagaagctgagaaactggtga-30 50 -acatcatcagcaagccaggt-30 #82 (cat. No. 04689054001)
SCD5
Forward primer Reverse primer Universal probe
50 -gacctgcttgctgatcctgt-30 50 -ccattcacgaagcatctcac-30 #34 (cat. No. 04687671001)
PLIN2
Forward primer Reverse primer Universal probe
50 -tcagctccattctactgttcacc-30 50 -cctgaattttctgattggcact-30 #72 (cat. No. 04688953001)
PPIA
Forward primer Reverse primer Universal probe
50 -cctaaagcatacgggtcctg-30 50 -tttcactttgccaaacacca-30 #48 (cat. No. 04688082001)
2. Materials and methods 2.1. Cell culture
Lipofectamine RNAiMAX (Invitrogen, Darmstadt, Germany) was used to transfect SZ95 sebocytes at 40% confluence in 6-well or 96-well plates with siRNAs for EGFR, ERBB2, ERBB3 or with a negative control siRNA (Silencer Select, Ambion, Austin, TX, USA). 24 h after transfection, cells were induced to synthesize lipids by adding LA to the culture medium as described above. The cells were allowed to differentiate for 48 h, harvested with ice-cold PBS and stored as cell pellets at 80 °C for the lipid analysis or dissolved in protein lysis buffer for Western blot analysis. 2.3. Quantitative RT-PCR Total RNA was isolated with TRIzol reagent (Invitrogen) and 1 lg of RNA samples were reverse-transcribed in a final volume of 20 ll using RevertAid Reverse Transcriptase (Thermo Scientific, Schwerte, Germany) according to the manufacturer’s instructions. Quantitative RT-PCR was carried out in a LightCyclerÒ 480 (Roche, Mannheim, Germany) using the primers listed in Table 1 (0.5 lM), 1 ll cDNA, 0.2 lM probe (Universal ProbeLibrary Set, Roche), and the LightCyclerÒ 480 Probes Master (Roche) in a final volume of 10 ll. Cycle conditions were 95 °C for 5 min for the first cycle, followed by 45 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 1 s. Transcript copy numbers were normalized to peptidylprolyl isomerase A (PPIA) mRNA copies. The DCt value of the sample was determined by subtracting the average Ct value of the target gene from the average Ct value of the PPIA gene. For each primer pair we performed no-template and no-RT control assays, which produced negligible signals that were usually greater than 40 in Ct value. Experiments were performed in duplicates for each sample. 2.4. Western blot analysis Protein was extracted using Laemmli-extraction-buffer, and the protein concentration was estimated via bicinchoninic acid protein assay. 30 lg of total protein were separated by 12% SDS–PAGE and transferred to PVDF membranes (Millipore, Schwalbach, Germany) by semidry blotting. Membranes were blocked in 5% w/v fat-free milk powder (Roth, Karlsruhe, Germany) for 1 h at room temperature. After washing in Tris-buffered saline solution with 1% Tween20 (Sigma, Taufkirchen, Germany), membranes were incubated over night at 4 °C in 5% w/v BSA (Sigma) with the appropriated primary antibody. Primary antibodies were rabbit anti-EGFR (1005), rabbit anti-ERBB2 (C-18), rabbit anti-ERBB3 (C-17)
(1:500; Santa Cruz, Heidelberg, Germany) and rabbit anti-A Tubulin (1:5000) (Cell Signaling, Frankfurt, Germany, #2125). After washing, membranes were incubated in 5% w/v fat-free milk powder with a horseradish peroxidase-labeled secondary antibody donkey anti-rabbit (1:2000; NA934V, GE Healthcare, Munich, Germany). Signals were detected using an enhanced chemiluminescence detection reagent (GE Healthcare) and appropriated X-ray films (GE Healthcare).
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2.5. Lipid analysis
120–1200. The molecular features extraction algorithm was used to extract individual molecular species by their accurate mass detected with the ToF MS. Lists of molecular features, which were the detected species characterized by accurate mass, isotopic pattern and absolute abundance, were produced from each analyzed sample and converted into exchange files then processed with Mass Profiler Professional 12 (MPP 12, see below). Molecular features shared by the analyzed samples were aligned by their retention time (RT) in the chromatographic run, and accurate mass axis in order to compare their expression across the different experimental conditions. Compounds detected in the different samples and presenting consistent RT (shift below 6% of the RT) and accurate mass (mass error below 6 ppm) were assigned as the same molecular species. Relative abundance of individual features was obtained by normalizing their peak area by the area of the ISTDs. Compound identification and annotation were performed on the basis of in house databases, built with previously collected MS data and using the METLIN Personal Metabolite Database by means of the IDbrowser tool, and the Molecular Formula Generator algorithm. The Human Metabolome Database (http://www.hmdb.ca/) and LIPIDMAPS (http://www.lipidmaps.org/) were used to confirm and extend the identification. Equipment, data and statistical analysis packages were from Agilent Technologies, CA, USA.
For the Nile red assay, SZ95 sebocytes were washed twice with PBS, stained for 10 min with DAPI (2.5 lg/ml, Sigma), washed once with PBS and then stained with AdipoRed (Lonza, Walkersville, MD, USA) according to the manufacturer’s instructions. After 10 min, the fluorescence (488 nm) was measured and quantified using the Operetta high content screening system (Perkin Elmer, Waltham, MA). Lipid composition of sebocyte cells was investigated by analyzing crude lipid extracts with rapid resolution-reversed phase–high performance liquid chromatography coupled with electrospray ionization and time of flight mass spectrometry (RR-RP/HPLCESI–ToF/MS) as previously described [19]. Briefly, lipids were extracted from 2 106 SZ95 sebocytes for each sample with absolute ethanol containing 0.025% buthylated hydroxytoluene (BHT, Sigma–Aldrich, St. Louis, MO, USA) to prevent autoxidation. N-lauroyl-D-erythro-sphingosylphosphorylcholine (SM 12:0, Avanti Polar Lipids Inc., Alabaster, AL, USA), deuterated cholesterol2,2,3,4,4,6-d6 (d6CH), and glyceryl-d5-trihexadecanoate (d5TG 48:0) (CDN isotopes Inc., Pointe-Claire, Quebec, Canada) were added as the internal standards (ISTDs) at a final concentration of 5, 50 and 10 lM, respectively, to control recovery of lipids, analytical performance and to calculate the relative abundance of detected lipids. The concentrated ethanol phase was extracted with ethyl acetate. Profiles of intact cell lipids were acquired in positive ion mode. Two experiments performed in duplicate were analyzed. Accurate mass spectra were acquired in the m/z mass range 2.0
fold expression
0.4 0.3 0.2 0.1 0.0
EGFR
C
Ct values of gene targets of qRT-PCR analysis were shown relative to Ct values of the PPIA cDNA in Fig. 1A and D. In
B
0.5
relative expression
A
2.6. Data pretreatment and statistical analysis
ERBB3
1.5
**
1.0
** 0.5
***
0.0
ERBB4
EGFR
ERBB2
ERBB3
D
LA
0.06
relative expression
Vehicle
ERBB2
Vehicle LA
EGFR ERBB2 ERBB3 TUB1A1
0.05 0.04 0.03 0.02 0.01 0.00
TGFA AREG EREG HBEGF EGF
E
Vehicle LA
fold expression
4
BTC EPGN
**
3
**
**
2 1
*** 0
TGFA
AREG
EREG
HBEGF
EGF
BTC
EPGN
Fig. 1. Abundance and regulation of EGFR/ERBB family members in SZ95 sebocytes. (A) Analysis of receptor transcript levels by quantitative RT-PCR in undifferentiated cells (n = 5 samples/group). (B) Expression of the receptors in SZ95 sebocytes treated with vehicle or LA for 48 h (n = 5 samples/group). (C) Western blot analysis of EGFR, ERBB2 and PLIN3 in undifferentiated cells (vehicle) and cells induced to differentiate by culture in LA-supplemented medium for 48 h. The blots were stripped and employed for the detection of tubulin as a loading control. (D) Analysis of EGFR ligand transcript levels by quantitative RT-PCR in undifferentiated cells (n = 5 samples/group). (E) Expression of EGFR ligands in sebocytes treated with vehicle or LA for 48 h (n = 5 samples/group). Data were analyzed by Student’s t-test. Asterisks indicate significant differences between vehicle and LA-treated sebocytes (⁄⁄P < 0.01, ⁄⁄⁄P < 0.001).
1379
EGFR
ERBB2
ERBB3
TUBA1A
TUBA1A
TUBA1A
1.5 1.0
**
0.5
ERBB2 siRNA neg. siRNA
2.0 1.5 1.0
* 0.5
ERBB3 siRNA
ERBB3 siRNA neg. siRNA
3
Fold expression
EGFR siRNA neg. siRNA
2.0
Fold expression
Fold expression
B
Control siRNA
No siRNA
ERBB2 siRNA
Control siRNA
No siRNA
EGFR siRNA
No siRNA
A
Control siRNA
M. Dahlhoff et al. / FEBS Letters 589 (2015) 1376–1382
2
*** ***
1
* 0.0 EGFR
ERBB2
0.0
ERBB3
EGFR
ERBB2
0
ERBB3
EGFR
ERBB2
ERBB3
Fig. 2. Downregulation of EGFR, ERBB2 and ERBB3 by siRNA transfection. (A) Western blot analysis showing the downregulation of the receptors following transfection of SZ95 sebocytes with specific siRNAs. (B) Quantitative RT-PCR revealed a statistically significant reduction in receptor transcript level following transfection with the corresponding siRNA (n = 5 replicates/group). Note the concomitant upregulation of EGFR and ERBB2 in ERBB3-depleted cells. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.
60
B
**
*
40 20
13 1
4 16 200
11
ERBB2
1
11
ERBB3
0 Control
EGFR
ERBB2
ERBB3
Control
EGFR
ERBB2
ERBB3
2.0
*
* Fold change
EGFR
400
0
D
C
600
Fluorescence intensity
80
Fluorescence intensity
A
*
1.5
*
*
*
* * *
*
EGFR siRNA ERBB2 siRNA ERBB3 siRNA
*
1.0 0.5 0.0 TG 44
E
TG 46
TG 48
TG 50
TG 52
TG 54
TG 56
TG 58
**
*
***
*
* *** ** * * *
CE 20:4
* *
** ** ** *
CE 20:3
**
CE 18:2
* 2
CE 18:0
**
3
** ** * * *** ** ** ** * **
CE 24:1
CE 22:6
CE 22:5
CE 22:4
CE 22:3
CE 22:2
CE 22:1
CE 20:5
CE 20:2
CE 20:1
CE 18:3
CE 18:1
CE 16:2
0
CE 16:1
1
CE 16:0
Fold change
4
Fig. 3. Downregulation of EGFR/ERBB impairs sebaceous lipogenesis. Nile red staining and quantification of the emitted fluorescence demonstrates increased lipid accumulation in vehicle-treated (A) SZ95 sebocytes with siRNA-mediated downregulation of EGFR and ERBB3, while no changes were observed in LA-treated cells (B). The results (n = 6 replicates/group) are representative for two independent experiments. (C) Venn’s diagram showing the number of entities that were differently or commonly regulated by downregulation of EGFR, ERBB2 or ERBB3. (D) Effects of EGFR, ERBB2, or ERBB3 downregulation on the relative amount of triglycerides (TGs). (E) Effects of EGFR, ERBB2, or ERBB3 downregulation on the relative amount of cholesteryl esters (CEs). (D and E) shows the average fold change of the annotated lipids relative to the control siRNA. *P < 0.05, **P < 0.01, ***P < 0.001.
Figs. 1B, E, 2B and 4, all values were related to the mean value of the undifferentiated cells and compared by Student’s t-test (GraphPad Prism version 5.0 for Windows, GraphPad Software,
San Diego, CA, USA). Data are presented as means ± S.D. or box-plots with median. Group differences were considered to be statistically significant if P < 0.05. Pretreatment of analytical data
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Fold expression
A
M. Dahlhoff et al. / FEBS Letters 589 (2015) 1376–1382
***
3
neg. siRNA
3.2. Downregulation of EGFR/ERBB has receptor-specific effects on sebaceous lipogenesis in SZ95 sebocytes
EGFR siRNA
**
2
1
0 SREBF1
Fold expression
B
FAS
3
*
SCD1
SCD5
neg. siRNA
FADS2
DGAT2
ERBB2 siRNA
**
2
PLIN2
** ***
**
1
0 SREBF1
Fold expression
C
4
***
FAS
***
SCD1
neg. siRNA
3
*
2
SCD5
***
FADS2
DGAT2
PLIN2
ERBB3 siRNA
**
*
*
DGAT2
PLIN2
1 0 SREBF1
FAS
SCD1
SCD5
FADS2
Fig. 4. Quantification of the transcript levels of the indicated markers of sebaceous lipogenesis by quantitative RT-PCR (expressed as changes relative to controls). N = 5 mice/genotype, ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 (Mann–Whitney U-test).
consisted of RT and m/z alignment and normalization by ISTDs, baselining toward blank runs to remove background noise, and logarithmic transformation of normalized abundance. Statistical evaluation of the aligned and normalized HPLC–ToF/MS data was performed using univariate analysis, including Student t-test and one-way analysis of variance (ANOVA) using the MPP 12 package. 3. Results 3.1. Expression of EGFR/ERBB receptors and EGFR ligands in sebocytes We employed quantitative RT-PCR to characterize the expression of the EGFR/ERBB system in SZ95 sebocytes, a widely employed sebocyte cell line [18]. Among the receptors, EGFR showed the highest expression level, ERBB2 and ERBB3 transcript levels was considerably less frequent, and no ERBB4 expression could be detected (Fig. 1A). To evaluate relative changes in receptor expression during differentiation, we treated SZ95 sebocytes with the essential fatty acid LA for 48 h, an efficient method to induce sebaceous lipogenesis. As shown in Fig. 1B, LA treatment significantly reduced the transcript levels of all three receptors, and this effect was confirmed, at least for ERBB2 and ERBB3, at the protein level (Fig. 1C). The evaluation of the transcript levels of the seven EGFR ligands [20] by qRT-PCR demonstrated that TGFA was the most abundantly expressed ligand, followed by AREG, EREG, HBEGF, EGF, BTC, and EPGN (Fig. 1D). Treatment with LA resulted in the upregulation of AREG, HBEGF, and EPGN levels, reduced BTC levels, and unchanged transcript abundance of TGFA, EREG, and EGF (Fig. 1E).
We employed siRNA-mediated downregulation to assess the importance of EGFR/ERBB receptors during sebaceous lipogenesis. Transfection of SZ95 sebocytes with specific siRNAs resulted in almost complete depletion of the corresponding receptor, in contrast to untreated cells or cells transfected with a negative control siRNA (Fig. 2A). Quantitative RT-PCR confirmed a statistically significant, substantial reduction in the transcript levels of the corresponding receptor (Fig. 2B). Interestingly, downregulation of ERBB3 was accompanied by an upregulation of EGFR and ERBB2, possibly as a compensatory mechanism (Fig. 2B). Next, the Operetta system was employed for quantifying the total lipid amount in Nile red-stained cells. In vehicle-treated SZ95 sebocytes, downregulation of EGFR and ERBB3 resulted in a significant increase in the total cellular lipid amount, while downregulation of ERBB2 did not affect this parameter (Fig. 3A). In LAtreated cells, in contrast, no changes in the lipid amount was observed, independently of the siRNA employed (Fig. 3B). To investigate in greater detail the impact of EGFR/ERBB downregulation on the lipid composition of vehicle-treated SZ95 sebocytes, we employed a method based on rapid resolution reversed phase high performance liquid chromatography coupled to electrospray–time of flight mass spectrometry (RR-RP/HPLC-ESI–ToF/MS), which allows for the simultaneous detection of several hundreds of neutral lipid species in their intact form. As shown in Fig. 3C, a total of 57 species were found to be modified in the EGFR/ERBB siRNAtreated samples compared to the control siRNA-treated samples. Furthermore, 16 samples were significantly modified in common by EGFR, ERBB2 or ERBB3. Downregulation of EGFR and ERBB3 resulted in the induction of several specific triglycerides (TGs), particularly of those bearing a total of 48–58 carbon atoms in the side fatty acids (Fig. 3D). ERBB2 downregulation did not affect TG abundance, thus confirming the Nile red data. In contrast, ERBB2 downregulation had a prominent effect in inducing the relative amount of cholesteryl esters (CEs), although CE species were also increased in SZ95 sebocytes treated with EGFR or ERBB3 siRNAs (Fig. 3E). Cholesterol levels were not modified in any of the studied conditions (data not shown). 3.3. Downregulation of EGFR/ERBB differentially affects the expression of sebaceous lipogenesis markers in SZ95 sebocytes Next, we assessed by RT-qPCR whether loss of EGFR/ERBB receptors affected the expression of key markers of the intracellular pathways that govern lipogenic activity. Transcripts encoding sterol regulatory element-binding protein-1 (SREBF1), a transcription factor that regulates numerous genes involved in lipid biosynthesis were found significantly upregulated following downregulation of EGFR, ERBB2, and ERBB3 (Fig. 4A–C). Consistently, expression of the FAS, which is a SREBF1 target gene, was significantly upregulated (Fig. 4A–C). The expression of the other examined genes was unchanged in EGFR-downregulated cells (Fig. 4A). We detected a reduction in the transcript levels of FADS2 and DGAT2, concomitantly to an increase in PLIN2 transcript levels in SZ95 cells in which ERBB2 was downregulated (Fig. 4B). In ERBB3-depleted cells, we observed an upregulation of all examined markers (Fig. 4C). 4. Discussion Using the SZ95 cell line, we showed that sebocytes express EGFR, ERBB2 and ERBB3, while no ERBB4 expression was detected. This is in agreement to previous reports that ERBB4 expression is
M. Dahlhoff et al. / FEBS Letters 589 (2015) 1376–1382
not detectable in human [21,22] or mouse [9] epidermis, and also not in human keratinocyte cell lines such as HaCaT [23] and A431 [24]. Notably, expression of all receptors was downregulated when lipogenesis was induced in SZ95 cells by adding LA to the culture medium. Changes in EGFR expression during keratinocyte proliferation and differentiation have been previously documented in a number of studies (reviewed in [25]), and it has been also shown that ERBB2 and ERBB3 expression is modulated during keratinocyte differentiation [26]. Similarly, EGFR ligand expression is affected by EGFR activation [27] and by induction of differentiation [22] in keratinocytes. Furthermore, in agreement to our data, it was shown that EGFR and ERBB2 are downregulated during the differentiation of preadipocytes [28]. Furthermore, Nile red staining and quantitative analysis of the emitted fluorescence revealed that siRNA-mediated downregulation of EGFR and ERBB3 in vehicletreated SZ95 sebocytes significantly increase the total cellular lipid amount. This observation, allied to the downregulation of EGFR/ ERBBs in LA-treated cells, suggest that EGFR signaling normally inhibits sebaceous lipogenesis. As SZ95 cells express significant amounts of EGFR ligands such as TGFA, AREG, and EREG, tonic autocrine receptor activation is likely to occur in vitro. While autocrine EGFR activation is also likely to happen in SG in the skin, EGFR ligands may also stem from other cell types as fibroblasts or keratinocytes in vivo. Notably, the epidermis is a rich source of different EGFR ligands, and their experimental loss or overabundance is associated with a variety of pathological changes [25]. In contrast, no changes in the lipid amount were observed, independently of the siRNA employed, in LA-treated cells. The latter observation may indicate that LA treatment represents a very potent stimulus that can hardly be influenced by downregulation of a single EGFR/ERBB family member. The effects of EGFR/ERBB downregulation on the lipid accumulation in vehicle-treated SZ95 sebocytes were largely confirmed by a spectrometry-based method: Downregulation of EGFR and ERBB3 resulted in the induction of several specific TGs, particularly of those bearing 48–58 carbon atoms in the side fatty acids, while ERBB2 downregulation did not affect TG abundance. Since TG represent 50% of sebaceous lipids [4,29], these changes are important for both total sebaceous lipids and lipid composition. In contrast, EGFR, ERBB2 and ERBB3 downregulation induced the relative amount of CEs. These effects were accompanied by receptor type-specific changes in the expression of sebocyte lipogenesis markers. Significant upregulation of SREBF1 and its target gene FAS was a common feature of the EGFR/ERBB downregulation. SREBF1, a transcription factor of the SREBP family [30], is known to play a crucial role in the lipogenic stimulus in sebocytes [31]. One of the primary mechanisms of the lipid synthesis regulation by SREBF1 is the induction of fatty acid synthesis and desaturation through FAS and SCD1 expression [32,33]. Expression levels of transcripts for enzymes involved in the biotransformation of FAs, mainly desaturases, and in the lipid storage were differently regulated following EGFR/ERBB downmodulation. The effects of the ERBB2 siRNA on the expression of FADS2 and PLIN2, an enzyme that catalyses the formation of sapienic acid [34] and a lipid droplet-associated protein that protects cells from lipolysis [19], respectively, remain to be explained. In ERBB3-depleted cells, we observed an upregulation of DGAT2, FADS2, PLIN2, SCD1 and SCD5. These extensive changes are compatible with the induction of TG and CE synthesis after ERBB3 downregulation. Alternatively, they may represent a consequence of the observed compensatory upregulation of EGFR and ERBB2 transcripts in ERBB3 siRNA-treated cells, and therefore an experimental artefact. In contrast, a reduction in the transcript levels of DGAT2 and FADS2, concomitantly to an increase in PLIN2 transcript levels were observed in ERBB2-downregulated SZ95 sebocytes. As DGAT2 is an essential enzyme for TG synthesis [35], its downregulation
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may explain while downregulation of ERBB2 (in contrast to EGFR and ERBB3) did not enhance TG synthesis in SZ95 sebocytes. In summary, our study reveals that sebocytes abundantly express EGFR, with lower levels of ERBB2 and ERBB3. These receptors are downregulated following induction of lipid synthesis by addition of LA to the culture medium, thus supporting the concept that regulation of EGFR/ERBB expression represents a mechanism for switching from a mitogenic to a differentiating phenotype. Importantly, siRNA-mediated downregulation revealed specific effects of these receptors in the accumulation of TGs and CEs, and in the expression of key markers of sebaceous lipid synthesis. Thus, EGFR and ERBB2/3 may represent attractive targets for modulating sebaceous lipogenesis, a key factor in the pathogenesis of sebaceous gland-associated skin diseases, such as acne. Conflict of interest The authors state no conflicts of interest. Acknowledgements The authors would like to thank Stefanie Riesemann (Gene Center, Munich) for technical support. E.C. and M.L. were supported by the Italian Ministry of Health through the Grant RF-2010-2316435. References [1] Jenkinson, D.M., Elder, H.Y., Montgomery, I. and Moss, V.A. (1985) Comparative studies of the ultrastructure of the sebaceous gland. Tissue Cell 17, 683–698. [2] Thody, A.J. and Shuster, S. (1989) Control and function of sebaceous glands. Physiol. Rev. 69, 383–416. [3] Schneider, M.R. and Paus, R. (2010) Sebocytes, multifaceted epithelial cells: lipid production and holocrine secretion. Int. J. Biochem. Cell Biol. 42, 181– 185. [4] Smith, K.R. and Thiboutot, D.M. (2008) Thematic review series: skin lipids. Sebaceous gland lipids: friend or foe? J. Lipid Res. 49, 271–281. [5] Zouboulis, C.C., Baron, J.M., Bohm, M., Kippenberger, S., Kurzen, H., Reichrath, J. and Thielitz, A. (2008) Frontiers in sebaceous gland biology and pathology. Exp. Dermatol. 17, 542–551. [6] Webster, G.F. (2002) Acne vulgaris. BMJ 325, 475–479. [7] Kurokawa, I., Danby, F.W., Ju, Q., Wang, X., Xiang, L.F., Xia, L., Chen, W., Nagy, I., Picardo, M., Suh, D.H., Ganceviciene, R., Schagen, S., Tsatsou, F. and Zouboulis, C.C. (2009) New developments in our understanding of acne pathogenesis and treatment. Exp. Dermatol. 18, 821–832. [8] Zouboulis, C.C., Jourdan, E. and Picardo, M. (2014) Acne is an inflammatory disease and alterations of sebum composition initiate acne lesions. J. Eur. Acad. Dermatol. Venereol. 28, 527–532. [9] Kiguchi, K., Bol, D., Carbajal, S., Beltran, L., Moats, S., Chan, K., Jorcano, J. and Digiovanni, J. (2000) Constitutive expression of erbB2 in epidermis of transgenic mice results in epidermal hyperproliferation and spontaneous skin tumor development. Oncogene 19, 4243–4254. [10] Nanney, L.B., Magid, M., Stoscheck, C.M. and King Jr., L.E. (1984) Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages. J. Invest Dermatol. 83, 385–393. [11] Akimoto, N., Sato, T., Sakiguchi, T., Kitamura, K., Kohno, Y. and Ito, A. (2002) Cell proliferation and lipid formation in hamster sebaceous gland cells. Dermatology 204, 118–123. [12] Guy, R., Ridden, C. and Kealey, T. (1996) The improved organ maintenance of the human sebaceous gland: modeling in vitro the effects of epidermal growth factor, androgens, estrogens, 13-cis retinoic acid, and phenol red. J. Invest Dermatol. 106, 454–460. [13] Zouboulis, C.C., Xia, L., Akamatsu, H., Seltmann, H., Fritsch, M., Hornemann, S., Ruhl, R., Chen, W., Nau, H. and Orfanos, C.E. (1998) The human sebocyte culture model provides new insights into development and management of seborrhoea and acne. Dermatology 196, 21–31. [14] Dahlhoff, M., Muller, A.K., Wolf, E., Werner, S. and Schneider, M.R. (2010) Epigen transgenic mice develop enlarged sebaceous glands. J. Invest Dermatol. 130, 623–626. [15] Dahlhoff, M., de Angelis, M.H., Wolf, E. and Schneider, M.R. (2013) Ligandindependent epidermal growth factor receptor hyperactivation increases sebaceous gland size and sebum secretion in mice. Exp. Dermatol. 22, 667– 669. [16] Yarden, Y. and Sliwkowski, M.X. (2001) Untangling the ErbB signalling network. Nat. Rev. Mol. Cell Biol. 2, 127–137. [17] Citri, A. and Yarden, Y. (2006) EGF-ERBB signalling: towards the systems level. Nat. Rev. Mol. Cell Biol. 7, 505–516.
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[18] Zouboulis, C.C., Seltmann, H., Neitzel, H. and Orfanos, C.E. (1999) Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95). J. Invest Dermatol. 113, 1011–1020. [19] Dahlhoff, M., Camera, E., Picardo, M., Zouboulis, C.C., Chan, L., Chang, B.H. and Schneider, M.R. (2013) PLIN2, the major perilipin regulated during sebocyte differentiation, controls sebaceous lipid accumulation in vitro and sebaceous gland size in vivo. Biochim. Biophys. Acta 1830, 4642–4649. [20] Schneider, M.R. and Wolf, E. (2009) The epidermal growth factor receptor ligands at a glance. J. Cell. Physiol. 218, 460–466. [21] Stoll, S.W., Kansra, S., Peshick, S., Fry, D.W., Leopold, W.R., Wiesen, J.F., Sibilia, M., Zhang, T., Werb, Z., Derynck, R., Wagner, E.F. and Elder, J.T. (2001) Differential utilization and localization of ErbB receptor tyrosine kinases in skin compared to normal and malignant keratinocytes. Neoplasia 3, 339–350. [22] Piepkorn, M., Predd, H., Underwood, R. and Cook, P. (2003) Proliferationdifferentiation relationships in the expression of heparin-binding epidermal growth factor-related factors and erbB receptors by normal and psoriatic human keratinocytes. Arch. Dermatol. Res. 295, 93–101. [23] Marques, M.M., Martinez, N., Rodriguez-Garcia, I. and Alonso, A. (1999) EGFR family-mediated signal transduction in the human keratinocyte cell line HaCaT. Exp. Cell Res. 252, 432–438. [24] Bowers, G., Reardon, D., Hewitt, T., Dent, P., Mikkelsen, R.B., Valerie, K., Lammering, G., Amir, C. and Schmidt-Ullrich, R.K. (2001) The relative role of ErbB1-4 receptor tyrosine kinases in radiation signal transduction responses of human carcinoma cells. Oncogene 20, 1388–1397. [25] Schneider, M.R., Werner, S., Paus, R. and Wolf, E. (2008) Beyond wavy hairs: the epidermal growth factor receptor and its ligands in skin biology and pathology. Am. J. Pathol. 173, 14–24.
[26] De Potter, I.Y., Poumay, Y., Squillace, K.A. and Pittelkow, M.R. (2001) Human EGF receptor (HER) family and heregulin members are differentially expressed in epidermal keratinocytes and modulate differentiation. Exp. Cell Res. 271, 315–328. [27] Stoll, S.W. and Elder, J.T. (1999) Differential regulation of EGF-like growth factor genes in human keratinocytes. Biochem. Biophys. Res. Commun. 265, 214–221. [28] Pagano, E. and Calvo, J.C. (2003) ErbB2 and EGFR are downmodulated during the differentiation of 3T3-L1 preadipocytes. J. Cell. Biochem. 90, 561–572. [29] Picardo, M., Ottaviani, M., Camera, E. and Mastrofrancesco, A. (2009) Sebaceous gland lipids. Dermatoendocrinology 1, 68–71. [30] Shimano, H. (2009) SREBPs: physiology and pathophysiology of the SREBP family. FEBS J. 276, 616–621. [31] Smith, T.M., Gilliland, K., Clawson, G.A. and Thiboutot, D. (2008) IGF-1 induces SREBP-1 expression and lipogenesis in SEB-1 sebocytes via activation of the phosphoinositide 3-kinase/Akt pathway. J. Invest Dermatol. 128, 1286–1293. [32] Bennett, M.K., Lopez, J.M., Sanchez, H.B. and Osborne, T.F. (1995) Sterol regulation of fatty acid synthase promoter. Coordinate feedback regulation of two major lipid pathways. J. Biol. Chem. 270, 25578–25583. [33] Eberle, D., Hegarty, B., Bossard, P., Ferre, P. and Foufelle, F. (2004) SREBP transcription factors: master regulators of lipid homeostasis. Biochimie 86, 839–848. [34] Ge, L., Gordon, J.S., Hsuan, C., Stenn, K. and Prouty, S.M. (2003) Identification of the delta-6 desaturase of human sebaceous glands: expression and enzyme activity. J. Invest Dermatol. 120, 707–714. [35] Buhman, K.K., Chen, H.C. and Farese Jr., R.V. (2001) The enzymes of neutral lipid synthesis. J. Biol. Chem. 276, 40369–40372.
journal homepage: www.FEBSLetters.org
EGFR/ERBB receptors differentially modulate sebaceous lipogenesis Maik Dahlhoff a, Emanuela Camera b, Matteo Ludovici b, Mauro Picardo b, Udo Müller c, Heinrich Leonhardt c, Christos C. Zouboulis d, Marlon R. Schneider a,⇑ a
Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy c Human Biology and BioImaging, Department of Biology II, LMU Munich, Germany d Departments of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center, Dessau, Germany b
a r t i c l e
i n f o
Article history: Received 5 December 2014 Revised 12 February 2015 Accepted 4 April 2015 Available online 15 April 2015 Edited by Laszlo Nagy Keywords: Sebaceous gland Epidermal growth factor receptor ERBB Sebaceous lipogenesis
a b s t r a c t The roles of the epidermal growth factor receptor (EGFR) in sebaceous glands remain poorly explored. We show that human sebocytes express EGFR and lower levels of ERBB2 and ERBB3, all receptors being downregulated after the induction of lipid synthesis. Nile red staining showed that siRNA-mediated downregulation of EGFR or ERBB3 increases lipid accumulation, whereas ERBB2 downregulation has no effect. Spectrometry confirmed induction of triglycerides after EGFR or ERBB3 downregulation and revealed induction of cholesteryl esters after downregulation of EGFR, ERBB2 or ERBB3. Thus, EGFR/ERBB receptors differentially modulate sebaceous lipogenesis, a key feature of sebaceous gland physiology and of several skin diseases. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Sebaceous glands (SG) are exocrine glands of the skin most commonly found in association with a hair follicle. During their differentiation, sebocytes progressively synthesize and accumulate lipids within their cytoplasm, and are eventually dislodged from the periphery to the core of the gland. Once a certain size is attained, the cell disrupts and releases its contents, a process termed holocrine secretion. The synthesized lipids and cellular debris reach the skin surface via the hair follicle canal, and represent the major component of skin sebum [1–3]. Although several functions have been credited to sebum, including a role in epidermal barrier and in hair follicle integrity, as well as antibacterial and antioxidant properties [4,5], its exact role remains unclear. However, increased sebum production is a key component the most common skin disease, acne [6,7]. Current data indicate that not only the amount of sebum but also the sebum composition plays a major Author’s contributions: Conception and design: M. Dahlhoff, E. Camera, M.R. Schneider. Acquisition of data: M. Dahlhoff, E. Camera, M. Ludovici, U. Müller, M.R. Schneider. Contribution of essential reagents: M. Picardo, H. Leonhardt, C.C. Zouboulis. Analysis and interpretation of data: M. Dahlhoff, E. Camera, M.R. Schneider. Writing, review, and/or revision of the manuscript: M.R. Schneider. ⇑ Corresponding author at: Gene Center, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany. E-mail address: [email protected] (M.R. Schneider).
role in the induction of sebaceous gland-associated skin diseases [8]. Sebocyte differentiation and sebaceous lipogenesis are controlled by numerous hormones, growth factors, and transcription factors. There is growing evidence suggesting that the epidermal growth factor receptor (EGFR) and its ligands play an important role in this process. The EGFR is strongly expressed in the undifferentiated sebocytes at the periphery of mouse [9] and human [10] sebaceous glands. Stimulation of hamster sebocytes with EGFR ligands increased proliferation and inhibited sebaceous lipogenesis [11], and the ligand EGF also impaired lipogenesis in human sebaceous glands maintained in vitro [12]. Interestingly, although EGF is a common medium additive for the culture of isolated human sebocytes, it does not influence their proliferation [13], which points to species-specific differences in the response of sebocytes to EGF. More recently, enlarged SGs and increased sebum production were reported in transgenic mice overexpressing the EGFR ligand epigen [14] and in Dsk5 animals, a mouse line in which the EGFR is constitutively activated independently of ligand binding [15]. The EGFR (also known as ERBB1 or HER1) belongs to a family of tyrosine kinase receptors that also includes ERBB2 (NEU, HER2), ERBB3 (HER3), and ERBB4 (HER4) [16,17]. In contrast to the EGFR, the expression and function of these related receptors in sebocytes has not been studied in detail before. Notably, a marked
http://dx.doi.org/10.1016/j.febslet.2015.04.003 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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enlargement of sebaceous glands was also reported in mice overexpressing ERBB2 [9], suggesting that ERBBs may be able to modulate sebocyte differentiation as well. Thus, the aim of this work is to study the expression and function of the ERBB receptors in sebocytes to uncover their potential functions in the biology of sebaceous glands.
Table 1 Primers and probes employed for the quantitative RT-PCR analysis. EGFR
Forward primer Reverse primer Universal probe
50 -gccttgactgaggacagca-30 50 -tttgggaacggactggttta-30 #69 (cat. No. 04688686001)
ERBB2
Forward primer Reverse primer Universal probe
50 -tgctgtcctgttcaccactc-30 50 -tcatcctcatcatcttcacattg-30 #67 (cat. No. 04688660001)
ERBB3
Forward primer Reverse primer Universal probe
50 -ctgatcaccggcctcaat-30 50 -ggaagacattgagcttctctgg-30 #37 (cat. No. 04687957001)
SZ95 sebocytes [18] were routinely cultured in SebomedÒ medium (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (Biochrom) and 5 lg/l EGF (Biochrom). For the experimental induction of lipogenesis, cells were cultured in the medium above lacking EGF for 24 h before supplementation with linoleic acid (LA; 104 M, Merck, Darmstadt, Germany) or vehicle only (DMSO) for the indicated periods of time.
ERBB4
Forward primer Reverse primer Universal probe
50 -gagcaagaattgactcgaatagg-30 50 -ttcctgacatgggggtgtag-30 #63 (cat. no. 04688627001)
TGFA
Forward primer Reverse primer Universal probe
50 -ttgctgccactcagaaacag-30 50 -atctgccacagtccacctg-30 #63 (cat. No. 04688627001)
AREG
Forward primer Reverse primer Universal probe
50 -cggagaatgcaaatatatagagcac-30 50 -caccgaaatattcttgctgaca-30 #38 (cat. No. 04687965001)
2.2. siRNA-mediated downregulation
EREG
Forward primer Reverse primer Universal probe
50 -tggtctcttcactcaggtctca-30 50 -cgtgagttggcatagggaac-30 #86 (cat. No. 04689119001)
HBEGF
Forward primer Reverse primer Universal probe
50 -tggggcttctcatgtttagg-30 50 -catgcccaacttcactttctc-30 #55 (cat. No. 04688520001)
EGF
Forward primer Reverse primer Universal probe
50 -aagaatgggggtcaaccagt-30 50 -tgaagttggttgcattgacc-30 #27 (cat. No. 04687582001)
BTC
Forward primer Reverse primer Universal probe
50 -actgcatcaaagggagatgc-30 50 -tctcacaccttgctccaatg-30 #49 (cat. No. 04688104001)
EPGN
Forward primer Reverse primer Universal probe
50 -tttgggagttccaatatcagc-30 50 -tgtgattggaggtgttacagtca-30 #34 (cat. No. 04687671001)
SREBF1
Forward primer Reverse primer Universal probe
5’-cgctcctccatcaatgaca-‘3 5’-tgcgcaagacagcagattta-‘3 #77 (cat. no. 04689003001)
FAS
Forward primer Reverse primer Universal probe
5’-gtggacccgctcagtacg-‘3 5’-ggacgataatctagcaacagacg-‘3 #60 (cat. no. 04688589001)
DGAT2
Forward primer Reverse primer Universal probe
5’-caagcccatcaccactgtt-30 50 -tcgatgtcttgctgggttg-30 #78 (cat. No. 04689011001)
FADS2
Forward primer Reverse primer Universal probe
50 -ctacgctggagaagatgcaa-30 50 -ttcaagaacttgcccacga-30 #85 (cat. No. 04689097001)
SCD1
Forward primer Reverse primer Universal probe
50 -cctagaagctgagaaactggtga-30 50 -acatcatcagcaagccaggt-30 #82 (cat. No. 04689054001)
SCD5
Forward primer Reverse primer Universal probe
50 -gacctgcttgctgatcctgt-30 50 -ccattcacgaagcatctcac-30 #34 (cat. No. 04687671001)
PLIN2
Forward primer Reverse primer Universal probe
50 -tcagctccattctactgttcacc-30 50 -cctgaattttctgattggcact-30 #72 (cat. No. 04688953001)
PPIA
Forward primer Reverse primer Universal probe
50 -cctaaagcatacgggtcctg-30 50 -tttcactttgccaaacacca-30 #48 (cat. No. 04688082001)
2. Materials and methods 2.1. Cell culture
Lipofectamine RNAiMAX (Invitrogen, Darmstadt, Germany) was used to transfect SZ95 sebocytes at 40% confluence in 6-well or 96-well plates with siRNAs for EGFR, ERBB2, ERBB3 or with a negative control siRNA (Silencer Select, Ambion, Austin, TX, USA). 24 h after transfection, cells were induced to synthesize lipids by adding LA to the culture medium as described above. The cells were allowed to differentiate for 48 h, harvested with ice-cold PBS and stored as cell pellets at 80 °C for the lipid analysis or dissolved in protein lysis buffer for Western blot analysis. 2.3. Quantitative RT-PCR Total RNA was isolated with TRIzol reagent (Invitrogen) and 1 lg of RNA samples were reverse-transcribed in a final volume of 20 ll using RevertAid Reverse Transcriptase (Thermo Scientific, Schwerte, Germany) according to the manufacturer’s instructions. Quantitative RT-PCR was carried out in a LightCyclerÒ 480 (Roche, Mannheim, Germany) using the primers listed in Table 1 (0.5 lM), 1 ll cDNA, 0.2 lM probe (Universal ProbeLibrary Set, Roche), and the LightCyclerÒ 480 Probes Master (Roche) in a final volume of 10 ll. Cycle conditions were 95 °C for 5 min for the first cycle, followed by 45 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 1 s. Transcript copy numbers were normalized to peptidylprolyl isomerase A (PPIA) mRNA copies. The DCt value of the sample was determined by subtracting the average Ct value of the target gene from the average Ct value of the PPIA gene. For each primer pair we performed no-template and no-RT control assays, which produced negligible signals that were usually greater than 40 in Ct value. Experiments were performed in duplicates for each sample. 2.4. Western blot analysis Protein was extracted using Laemmli-extraction-buffer, and the protein concentration was estimated via bicinchoninic acid protein assay. 30 lg of total protein were separated by 12% SDS–PAGE and transferred to PVDF membranes (Millipore, Schwalbach, Germany) by semidry blotting. Membranes were blocked in 5% w/v fat-free milk powder (Roth, Karlsruhe, Germany) for 1 h at room temperature. After washing in Tris-buffered saline solution with 1% Tween20 (Sigma, Taufkirchen, Germany), membranes were incubated over night at 4 °C in 5% w/v BSA (Sigma) with the appropriated primary antibody. Primary antibodies were rabbit anti-EGFR (1005), rabbit anti-ERBB2 (C-18), rabbit anti-ERBB3 (C-17)
(1:500; Santa Cruz, Heidelberg, Germany) and rabbit anti-A Tubulin (1:5000) (Cell Signaling, Frankfurt, Germany, #2125). After washing, membranes were incubated in 5% w/v fat-free milk powder with a horseradish peroxidase-labeled secondary antibody donkey anti-rabbit (1:2000; NA934V, GE Healthcare, Munich, Germany). Signals were detected using an enhanced chemiluminescence detection reagent (GE Healthcare) and appropriated X-ray films (GE Healthcare).
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2.5. Lipid analysis
120–1200. The molecular features extraction algorithm was used to extract individual molecular species by their accurate mass detected with the ToF MS. Lists of molecular features, which were the detected species characterized by accurate mass, isotopic pattern and absolute abundance, were produced from each analyzed sample and converted into exchange files then processed with Mass Profiler Professional 12 (MPP 12, see below). Molecular features shared by the analyzed samples were aligned by their retention time (RT) in the chromatographic run, and accurate mass axis in order to compare their expression across the different experimental conditions. Compounds detected in the different samples and presenting consistent RT (shift below 6% of the RT) and accurate mass (mass error below 6 ppm) were assigned as the same molecular species. Relative abundance of individual features was obtained by normalizing their peak area by the area of the ISTDs. Compound identification and annotation were performed on the basis of in house databases, built with previously collected MS data and using the METLIN Personal Metabolite Database by means of the IDbrowser tool, and the Molecular Formula Generator algorithm. The Human Metabolome Database (http://www.hmdb.ca/) and LIPIDMAPS (http://www.lipidmaps.org/) were used to confirm and extend the identification. Equipment, data and statistical analysis packages were from Agilent Technologies, CA, USA.
For the Nile red assay, SZ95 sebocytes were washed twice with PBS, stained for 10 min with DAPI (2.5 lg/ml, Sigma), washed once with PBS and then stained with AdipoRed (Lonza, Walkersville, MD, USA) according to the manufacturer’s instructions. After 10 min, the fluorescence (488 nm) was measured and quantified using the Operetta high content screening system (Perkin Elmer, Waltham, MA). Lipid composition of sebocyte cells was investigated by analyzing crude lipid extracts with rapid resolution-reversed phase–high performance liquid chromatography coupled with electrospray ionization and time of flight mass spectrometry (RR-RP/HPLCESI–ToF/MS) as previously described [19]. Briefly, lipids were extracted from 2 106 SZ95 sebocytes for each sample with absolute ethanol containing 0.025% buthylated hydroxytoluene (BHT, Sigma–Aldrich, St. Louis, MO, USA) to prevent autoxidation. N-lauroyl-D-erythro-sphingosylphosphorylcholine (SM 12:0, Avanti Polar Lipids Inc., Alabaster, AL, USA), deuterated cholesterol2,2,3,4,4,6-d6 (d6CH), and glyceryl-d5-trihexadecanoate (d5TG 48:0) (CDN isotopes Inc., Pointe-Claire, Quebec, Canada) were added as the internal standards (ISTDs) at a final concentration of 5, 50 and 10 lM, respectively, to control recovery of lipids, analytical performance and to calculate the relative abundance of detected lipids. The concentrated ethanol phase was extracted with ethyl acetate. Profiles of intact cell lipids were acquired in positive ion mode. Two experiments performed in duplicate were analyzed. Accurate mass spectra were acquired in the m/z mass range 2.0
fold expression
0.4 0.3 0.2 0.1 0.0
EGFR
C
Ct values of gene targets of qRT-PCR analysis were shown relative to Ct values of the PPIA cDNA in Fig. 1A and D. In
B
0.5
relative expression
A
2.6. Data pretreatment and statistical analysis
ERBB3
1.5
**
1.0
** 0.5
***
0.0
ERBB4
EGFR
ERBB2
ERBB3
D
LA
0.06
relative expression
Vehicle
ERBB2
Vehicle LA
EGFR ERBB2 ERBB3 TUB1A1
0.05 0.04 0.03 0.02 0.01 0.00
TGFA AREG EREG HBEGF EGF
E
Vehicle LA
fold expression
4
BTC EPGN
**
3
**
**
2 1
*** 0
TGFA
AREG
EREG
HBEGF
EGF
BTC
EPGN
Fig. 1. Abundance and regulation of EGFR/ERBB family members in SZ95 sebocytes. (A) Analysis of receptor transcript levels by quantitative RT-PCR in undifferentiated cells (n = 5 samples/group). (B) Expression of the receptors in SZ95 sebocytes treated with vehicle or LA for 48 h (n = 5 samples/group). (C) Western blot analysis of EGFR, ERBB2 and PLIN3 in undifferentiated cells (vehicle) and cells induced to differentiate by culture in LA-supplemented medium for 48 h. The blots were stripped and employed for the detection of tubulin as a loading control. (D) Analysis of EGFR ligand transcript levels by quantitative RT-PCR in undifferentiated cells (n = 5 samples/group). (E) Expression of EGFR ligands in sebocytes treated with vehicle or LA for 48 h (n = 5 samples/group). Data were analyzed by Student’s t-test. Asterisks indicate significant differences between vehicle and LA-treated sebocytes (⁄⁄P < 0.01, ⁄⁄⁄P < 0.001).
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EGFR
ERBB2
ERBB3
TUBA1A
TUBA1A
TUBA1A
1.5 1.0
**
0.5
ERBB2 siRNA neg. siRNA
2.0 1.5 1.0
* 0.5
ERBB3 siRNA
ERBB3 siRNA neg. siRNA
3
Fold expression
EGFR siRNA neg. siRNA
2.0
Fold expression
Fold expression
B
Control siRNA
No siRNA
ERBB2 siRNA
Control siRNA
No siRNA
EGFR siRNA
No siRNA
A
Control siRNA
M. Dahlhoff et al. / FEBS Letters 589 (2015) 1376–1382
2
*** ***
1
* 0.0 EGFR
ERBB2
0.0
ERBB3
EGFR
ERBB2
0
ERBB3
EGFR
ERBB2
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Fig. 2. Downregulation of EGFR, ERBB2 and ERBB3 by siRNA transfection. (A) Western blot analysis showing the downregulation of the receptors following transfection of SZ95 sebocytes with specific siRNAs. (B) Quantitative RT-PCR revealed a statistically significant reduction in receptor transcript level following transfection with the corresponding siRNA (n = 5 replicates/group). Note the concomitant upregulation of EGFR and ERBB2 in ERBB3-depleted cells. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.
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Fig. 3. Downregulation of EGFR/ERBB impairs sebaceous lipogenesis. Nile red staining and quantification of the emitted fluorescence demonstrates increased lipid accumulation in vehicle-treated (A) SZ95 sebocytes with siRNA-mediated downregulation of EGFR and ERBB3, while no changes were observed in LA-treated cells (B). The results (n = 6 replicates/group) are representative for two independent experiments. (C) Venn’s diagram showing the number of entities that were differently or commonly regulated by downregulation of EGFR, ERBB2 or ERBB3. (D) Effects of EGFR, ERBB2, or ERBB3 downregulation on the relative amount of triglycerides (TGs). (E) Effects of EGFR, ERBB2, or ERBB3 downregulation on the relative amount of cholesteryl esters (CEs). (D and E) shows the average fold change of the annotated lipids relative to the control siRNA. *P < 0.05, **P < 0.01, ***P < 0.001.
Figs. 1B, E, 2B and 4, all values were related to the mean value of the undifferentiated cells and compared by Student’s t-test (GraphPad Prism version 5.0 for Windows, GraphPad Software,
San Diego, CA, USA). Data are presented as means ± S.D. or box-plots with median. Group differences were considered to be statistically significant if P < 0.05. Pretreatment of analytical data
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Fig. 4. Quantification of the transcript levels of the indicated markers of sebaceous lipogenesis by quantitative RT-PCR (expressed as changes relative to controls). N = 5 mice/genotype, ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 (Mann–Whitney U-test).
consisted of RT and m/z alignment and normalization by ISTDs, baselining toward blank runs to remove background noise, and logarithmic transformation of normalized abundance. Statistical evaluation of the aligned and normalized HPLC–ToF/MS data was performed using univariate analysis, including Student t-test and one-way analysis of variance (ANOVA) using the MPP 12 package. 3. Results 3.1. Expression of EGFR/ERBB receptors and EGFR ligands in sebocytes We employed quantitative RT-PCR to characterize the expression of the EGFR/ERBB system in SZ95 sebocytes, a widely employed sebocyte cell line [18]. Among the receptors, EGFR showed the highest expression level, ERBB2 and ERBB3 transcript levels was considerably less frequent, and no ERBB4 expression could be detected (Fig. 1A). To evaluate relative changes in receptor expression during differentiation, we treated SZ95 sebocytes with the essential fatty acid LA for 48 h, an efficient method to induce sebaceous lipogenesis. As shown in Fig. 1B, LA treatment significantly reduced the transcript levels of all three receptors, and this effect was confirmed, at least for ERBB2 and ERBB3, at the protein level (Fig. 1C). The evaluation of the transcript levels of the seven EGFR ligands [20] by qRT-PCR demonstrated that TGFA was the most abundantly expressed ligand, followed by AREG, EREG, HBEGF, EGF, BTC, and EPGN (Fig. 1D). Treatment with LA resulted in the upregulation of AREG, HBEGF, and EPGN levels, reduced BTC levels, and unchanged transcript abundance of TGFA, EREG, and EGF (Fig. 1E).
We employed siRNA-mediated downregulation to assess the importance of EGFR/ERBB receptors during sebaceous lipogenesis. Transfection of SZ95 sebocytes with specific siRNAs resulted in almost complete depletion of the corresponding receptor, in contrast to untreated cells or cells transfected with a negative control siRNA (Fig. 2A). Quantitative RT-PCR confirmed a statistically significant, substantial reduction in the transcript levels of the corresponding receptor (Fig. 2B). Interestingly, downregulation of ERBB3 was accompanied by an upregulation of EGFR and ERBB2, possibly as a compensatory mechanism (Fig. 2B). Next, the Operetta system was employed for quantifying the total lipid amount in Nile red-stained cells. In vehicle-treated SZ95 sebocytes, downregulation of EGFR and ERBB3 resulted in a significant increase in the total cellular lipid amount, while downregulation of ERBB2 did not affect this parameter (Fig. 3A). In LAtreated cells, in contrast, no changes in the lipid amount was observed, independently of the siRNA employed (Fig. 3B). To investigate in greater detail the impact of EGFR/ERBB downregulation on the lipid composition of vehicle-treated SZ95 sebocytes, we employed a method based on rapid resolution reversed phase high performance liquid chromatography coupled to electrospray–time of flight mass spectrometry (RR-RP/HPLC-ESI–ToF/MS), which allows for the simultaneous detection of several hundreds of neutral lipid species in their intact form. As shown in Fig. 3C, a total of 57 species were found to be modified in the EGFR/ERBB siRNAtreated samples compared to the control siRNA-treated samples. Furthermore, 16 samples were significantly modified in common by EGFR, ERBB2 or ERBB3. Downregulation of EGFR and ERBB3 resulted in the induction of several specific triglycerides (TGs), particularly of those bearing a total of 48–58 carbon atoms in the side fatty acids (Fig. 3D). ERBB2 downregulation did not affect TG abundance, thus confirming the Nile red data. In contrast, ERBB2 downregulation had a prominent effect in inducing the relative amount of cholesteryl esters (CEs), although CE species were also increased in SZ95 sebocytes treated with EGFR or ERBB3 siRNAs (Fig. 3E). Cholesterol levels were not modified in any of the studied conditions (data not shown). 3.3. Downregulation of EGFR/ERBB differentially affects the expression of sebaceous lipogenesis markers in SZ95 sebocytes Next, we assessed by RT-qPCR whether loss of EGFR/ERBB receptors affected the expression of key markers of the intracellular pathways that govern lipogenic activity. Transcripts encoding sterol regulatory element-binding protein-1 (SREBF1), a transcription factor that regulates numerous genes involved in lipid biosynthesis were found significantly upregulated following downregulation of EGFR, ERBB2, and ERBB3 (Fig. 4A–C). Consistently, expression of the FAS, which is a SREBF1 target gene, was significantly upregulated (Fig. 4A–C). The expression of the other examined genes was unchanged in EGFR-downregulated cells (Fig. 4A). We detected a reduction in the transcript levels of FADS2 and DGAT2, concomitantly to an increase in PLIN2 transcript levels in SZ95 cells in which ERBB2 was downregulated (Fig. 4B). In ERBB3-depleted cells, we observed an upregulation of all examined markers (Fig. 4C). 4. Discussion Using the SZ95 cell line, we showed that sebocytes express EGFR, ERBB2 and ERBB3, while no ERBB4 expression was detected. This is in agreement to previous reports that ERBB4 expression is
M. Dahlhoff et al. / FEBS Letters 589 (2015) 1376–1382
not detectable in human [21,22] or mouse [9] epidermis, and also not in human keratinocyte cell lines such as HaCaT [23] and A431 [24]. Notably, expression of all receptors was downregulated when lipogenesis was induced in SZ95 cells by adding LA to the culture medium. Changes in EGFR expression during keratinocyte proliferation and differentiation have been previously documented in a number of studies (reviewed in [25]), and it has been also shown that ERBB2 and ERBB3 expression is modulated during keratinocyte differentiation [26]. Similarly, EGFR ligand expression is affected by EGFR activation [27] and by induction of differentiation [22] in keratinocytes. Furthermore, in agreement to our data, it was shown that EGFR and ERBB2 are downregulated during the differentiation of preadipocytes [28]. Furthermore, Nile red staining and quantitative analysis of the emitted fluorescence revealed that siRNA-mediated downregulation of EGFR and ERBB3 in vehicletreated SZ95 sebocytes significantly increase the total cellular lipid amount. This observation, allied to the downregulation of EGFR/ ERBBs in LA-treated cells, suggest that EGFR signaling normally inhibits sebaceous lipogenesis. As SZ95 cells express significant amounts of EGFR ligands such as TGFA, AREG, and EREG, tonic autocrine receptor activation is likely to occur in vitro. While autocrine EGFR activation is also likely to happen in SG in the skin, EGFR ligands may also stem from other cell types as fibroblasts or keratinocytes in vivo. Notably, the epidermis is a rich source of different EGFR ligands, and their experimental loss or overabundance is associated with a variety of pathological changes [25]. In contrast, no changes in the lipid amount were observed, independently of the siRNA employed, in LA-treated cells. The latter observation may indicate that LA treatment represents a very potent stimulus that can hardly be influenced by downregulation of a single EGFR/ERBB family member. The effects of EGFR/ERBB downregulation on the lipid accumulation in vehicle-treated SZ95 sebocytes were largely confirmed by a spectrometry-based method: Downregulation of EGFR and ERBB3 resulted in the induction of several specific TGs, particularly of those bearing 48–58 carbon atoms in the side fatty acids, while ERBB2 downregulation did not affect TG abundance. Since TG represent 50% of sebaceous lipids [4,29], these changes are important for both total sebaceous lipids and lipid composition. In contrast, EGFR, ERBB2 and ERBB3 downregulation induced the relative amount of CEs. These effects were accompanied by receptor type-specific changes in the expression of sebocyte lipogenesis markers. Significant upregulation of SREBF1 and its target gene FAS was a common feature of the EGFR/ERBB downregulation. SREBF1, a transcription factor of the SREBP family [30], is known to play a crucial role in the lipogenic stimulus in sebocytes [31]. One of the primary mechanisms of the lipid synthesis regulation by SREBF1 is the induction of fatty acid synthesis and desaturation through FAS and SCD1 expression [32,33]. Expression levels of transcripts for enzymes involved in the biotransformation of FAs, mainly desaturases, and in the lipid storage were differently regulated following EGFR/ERBB downmodulation. The effects of the ERBB2 siRNA on the expression of FADS2 and PLIN2, an enzyme that catalyses the formation of sapienic acid [34] and a lipid droplet-associated protein that protects cells from lipolysis [19], respectively, remain to be explained. In ERBB3-depleted cells, we observed an upregulation of DGAT2, FADS2, PLIN2, SCD1 and SCD5. These extensive changes are compatible with the induction of TG and CE synthesis after ERBB3 downregulation. Alternatively, they may represent a consequence of the observed compensatory upregulation of EGFR and ERBB2 transcripts in ERBB3 siRNA-treated cells, and therefore an experimental artefact. In contrast, a reduction in the transcript levels of DGAT2 and FADS2, concomitantly to an increase in PLIN2 transcript levels were observed in ERBB2-downregulated SZ95 sebocytes. As DGAT2 is an essential enzyme for TG synthesis [35], its downregulation
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may explain while downregulation of ERBB2 (in contrast to EGFR and ERBB3) did not enhance TG synthesis in SZ95 sebocytes. In summary, our study reveals that sebocytes abundantly express EGFR, with lower levels of ERBB2 and ERBB3. These receptors are downregulated following induction of lipid synthesis by addition of LA to the culture medium, thus supporting the concept that regulation of EGFR/ERBB expression represents a mechanism for switching from a mitogenic to a differentiating phenotype. Importantly, siRNA-mediated downregulation revealed specific effects of these receptors in the accumulation of TGs and CEs, and in the expression of key markers of sebaceous lipid synthesis. Thus, EGFR and ERBB2/3 may represent attractive targets for modulating sebaceous lipogenesis, a key factor in the pathogenesis of sebaceous gland-associated skin diseases, such as acne. Conflict of interest The authors state no conflicts of interest. Acknowledgements The authors would like to thank Stefanie Riesemann (Gene Center, Munich) for technical support. E.C. and M.L. were supported by the Italian Ministry of Health through the Grant RF-2010-2316435. References [1] Jenkinson, D.M., Elder, H.Y., Montgomery, I. and Moss, V.A. (1985) Comparative studies of the ultrastructure of the sebaceous gland. Tissue Cell 17, 683–698. [2] Thody, A.J. and Shuster, S. (1989) Control and function of sebaceous glands. Physiol. Rev. 69, 383–416. [3] Schneider, M.R. and Paus, R. (2010) Sebocytes, multifaceted epithelial cells: lipid production and holocrine secretion. Int. J. Biochem. Cell Biol. 42, 181– 185. [4] Smith, K.R. and Thiboutot, D.M. (2008) Thematic review series: skin lipids. Sebaceous gland lipids: friend or foe? J. Lipid Res. 49, 271–281. [5] Zouboulis, C.C., Baron, J.M., Bohm, M., Kippenberger, S., Kurzen, H., Reichrath, J. and Thielitz, A. (2008) Frontiers in sebaceous gland biology and pathology. Exp. Dermatol. 17, 542–551. [6] Webster, G.F. (2002) Acne vulgaris. BMJ 325, 475–479. [7] Kurokawa, I., Danby, F.W., Ju, Q., Wang, X., Xiang, L.F., Xia, L., Chen, W., Nagy, I., Picardo, M., Suh, D.H., Ganceviciene, R., Schagen, S., Tsatsou, F. and Zouboulis, C.C. (2009) New developments in our understanding of acne pathogenesis and treatment. Exp. Dermatol. 18, 821–832. [8] Zouboulis, C.C., Jourdan, E. and Picardo, M. (2014) Acne is an inflammatory disease and alterations of sebum composition initiate acne lesions. J. Eur. Acad. Dermatol. Venereol. 28, 527–532. [9] Kiguchi, K., Bol, D., Carbajal, S., Beltran, L., Moats, S., Chan, K., Jorcano, J. and Digiovanni, J. (2000) Constitutive expression of erbB2 in epidermis of transgenic mice results in epidermal hyperproliferation and spontaneous skin tumor development. Oncogene 19, 4243–4254. [10] Nanney, L.B., Magid, M., Stoscheck, C.M. and King Jr., L.E. (1984) Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages. J. Invest Dermatol. 83, 385–393. [11] Akimoto, N., Sato, T., Sakiguchi, T., Kitamura, K., Kohno, Y. and Ito, A. (2002) Cell proliferation and lipid formation in hamster sebaceous gland cells. Dermatology 204, 118–123. [12] Guy, R., Ridden, C. and Kealey, T. (1996) The improved organ maintenance of the human sebaceous gland: modeling in vitro the effects of epidermal growth factor, androgens, estrogens, 13-cis retinoic acid, and phenol red. J. Invest Dermatol. 106, 454–460. [13] Zouboulis, C.C., Xia, L., Akamatsu, H., Seltmann, H., Fritsch, M., Hornemann, S., Ruhl, R., Chen, W., Nau, H. and Orfanos, C.E. (1998) The human sebocyte culture model provides new insights into development and management of seborrhoea and acne. Dermatology 196, 21–31. [14] Dahlhoff, M., Muller, A.K., Wolf, E., Werner, S. and Schneider, M.R. (2010) Epigen transgenic mice develop enlarged sebaceous glands. J. Invest Dermatol. 130, 623–626. [15] Dahlhoff, M., de Angelis, M.H., Wolf, E. and Schneider, M.R. (2013) Ligandindependent epidermal growth factor receptor hyperactivation increases sebaceous gland size and sebum secretion in mice. Exp. Dermatol. 22, 667– 669. [16] Yarden, Y. and Sliwkowski, M.X. (2001) Untangling the ErbB signalling network. Nat. Rev. Mol. Cell Biol. 2, 127–137. [17] Citri, A. and Yarden, Y. (2006) EGF-ERBB signalling: towards the systems level. Nat. Rev. Mol. Cell Biol. 7, 505–516.
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[18] Zouboulis, C.C., Seltmann, H., Neitzel, H. and Orfanos, C.E. (1999) Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95). J. Invest Dermatol. 113, 1011–1020. [19] Dahlhoff, M., Camera, E., Picardo, M., Zouboulis, C.C., Chan, L., Chang, B.H. and Schneider, M.R. (2013) PLIN2, the major perilipin regulated during sebocyte differentiation, controls sebaceous lipid accumulation in vitro and sebaceous gland size in vivo. Biochim. Biophys. Acta 1830, 4642–4649. [20] Schneider, M.R. and Wolf, E. (2009) The epidermal growth factor receptor ligands at a glance. J. Cell. Physiol. 218, 460–466. [21] Stoll, S.W., Kansra, S., Peshick, S., Fry, D.W., Leopold, W.R., Wiesen, J.F., Sibilia, M., Zhang, T., Werb, Z., Derynck, R., Wagner, E.F. and Elder, J.T. (2001) Differential utilization and localization of ErbB receptor tyrosine kinases in skin compared to normal and malignant keratinocytes. Neoplasia 3, 339–350. [22] Piepkorn, M., Predd, H., Underwood, R. and Cook, P. (2003) Proliferationdifferentiation relationships in the expression of heparin-binding epidermal growth factor-related factors and erbB receptors by normal and psoriatic human keratinocytes. Arch. Dermatol. Res. 295, 93–101. [23] Marques, M.M., Martinez, N., Rodriguez-Garcia, I. and Alonso, A. (1999) EGFR family-mediated signal transduction in the human keratinocyte cell line HaCaT. Exp. Cell Res. 252, 432–438. [24] Bowers, G., Reardon, D., Hewitt, T., Dent, P., Mikkelsen, R.B., Valerie, K., Lammering, G., Amir, C. and Schmidt-Ullrich, R.K. (2001) The relative role of ErbB1-4 receptor tyrosine kinases in radiation signal transduction responses of human carcinoma cells. Oncogene 20, 1388–1397. [25] Schneider, M.R., Werner, S., Paus, R. and Wolf, E. (2008) Beyond wavy hairs: the epidermal growth factor receptor and its ligands in skin biology and pathology. Am. J. Pathol. 173, 14–24.
[26] De Potter, I.Y., Poumay, Y., Squillace, K.A. and Pittelkow, M.R. (2001) Human EGF receptor (HER) family and heregulin members are differentially expressed in epidermal keratinocytes and modulate differentiation. Exp. Cell Res. 271, 315–328. [27] Stoll, S.W. and Elder, J.T. (1999) Differential regulation of EGF-like growth factor genes in human keratinocytes. Biochem. Biophys. Res. Commun. 265, 214–221. [28] Pagano, E. and Calvo, J.C. (2003) ErbB2 and EGFR are downmodulated during the differentiation of 3T3-L1 preadipocytes. J. Cell. Biochem. 90, 561–572. [29] Picardo, M., Ottaviani, M., Camera, E. and Mastrofrancesco, A. (2009) Sebaceous gland lipids. Dermatoendocrinology 1, 68–71. [30] Shimano, H. (2009) SREBPs: physiology and pathophysiology of the SREBP family. FEBS J. 276, 616–621. [31] Smith, T.M., Gilliland, K., Clawson, G.A. and Thiboutot, D. (2008) IGF-1 induces SREBP-1 expression and lipogenesis in SEB-1 sebocytes via activation of the phosphoinositide 3-kinase/Akt pathway. J. Invest Dermatol. 128, 1286–1293. [32] Bennett, M.K., Lopez, J.M., Sanchez, H.B. and Osborne, T.F. (1995) Sterol regulation of fatty acid synthase promoter. Coordinate feedback regulation of two major lipid pathways. J. Biol. Chem. 270, 25578–25583. [33] Eberle, D., Hegarty, B., Bossard, P., Ferre, P. and Foufelle, F. (2004) SREBP transcription factors: master regulators of lipid homeostasis. Biochimie 86, 839–848. [34] Ge, L., Gordon, J.S., Hsuan, C., Stenn, K. and Prouty, S.M. (2003) Identification of the delta-6 desaturase of human sebaceous glands: expression and enzyme activity. J. Invest Dermatol. 120, 707–714. [35] Buhman, K.K., Chen, H.C. and Farese Jr., R.V. (2001) The enzymes of neutral lipid synthesis. J. Biol. Chem. 276, 40369–40372.
