Clin. exp. Immunol. (1990) 81, 97- 100
Elevated transcription of heat shock protein gene in scleroderma fibroblasts Y. DEGUCHI, N. SHIBATA* & S. KISHIMOTO Third Department of Internal Medicine, Osaka University School of Medicine, Osaka and * Department of Internal Medicine, Centre for Adult Diseases in Osaka, Osaka, Japan (Acceptedfor publication 22 January 1990)
SUMMARY Scleroderma is a systemic disorder characterized by fibrosis, which affects skin, lung, kidney and other organs. Heat shock protein (hsp) (70 kD) has been implicated as an essential element of cell function in cell growth and differentiation. To study the molecular basis of intracellular events in scleroderma fibroblasts, we compared the expression of hsp 70 gene in scleroderma and normal control fibroblasts by nuclear run on transcription assay and Northern blot assay. We show that scleroderma fibroblasts express more than eight times higher level of hsp 70 transcription in normal control fibroblasts at quiescent conditions in the absence of serum. After stimulation with serum, the transcription level of the hsp 70 gene is similar in scleroderma and normal control fibroblasts. Therefore, our results indicate an alteration/activation of intracellular events in scleroderma fibroblasts.
Keywords heat shock protein
gene
transcription scleroderma fibrosis
INTRODUCTION
In order to study the molecular basis of intracellular disorders in scleroderma fibroblasts, we examined hsp 70 gene expression in scleroderma and control fibroblasts.
In systemic autoimmune disorders such as scleroderma, connective tissue inflammation and microvascular damage resulting in multi-organ system involvement are the most prominent pathological changes. Scleroderma is a systemic disorder characterized by fibrosis and over-production of extracellular matrix components (Haynes & Gershwin, 1982). The mechanisms of fibrosis in scleroderma is not clearly known. Recently it was suggested that some population of fibroblasts in fibrotic lesion is changed and causes over-production of extracellular matrix (LeRoy et al., 1988; Trojanowska, Wu & LeRoy, 1988). Most of the intracellular disorders in sclerotic fibroblasts are still unknown. Changes of gene expression in response to heat shock stress have been described in human cells (Lindquist & Craig, 1988). The general effect of heat shock stress suppresses normal protein synthesis and enhances the synthesis of proteins such as heat shock proteins (hsp). Schlesinger, Aliperti & Kelly (1982) showed that various chemical, mechanical and environmental stresses could induce hsp; and the expression of hsp was regulated developmentally and in cell differentiation (Morange et al., 1984). It has been suggested that these proteins could be important for essential functions in cell growth and differentiation (Morange et al., 1984). The hsp of the 70 kD (hsp 70) family includes the inducible and cognate 68 and 72 kD hsp. Hsp 70 is the most conserved and well characterized.
MATERIALS AND METHODS
Skin biopsies and cell culture Skin fibroblasts were obtained by skin biopsy from affected portions of 15 scleroderma patients with diffuse cutaneous systemic sclerosis, as shown in Table 1. The diagnosis of scleroderma was made clinically in addition to the biopsy findings characterized by thinned epidermis with loss of rete pegs and a marked increase in collagen fibres within the dermis (Barnett, 1978). Control fibroblasts were obtained by forearm or pretibial skin biopsy of healthy donors who had no history of rheumatic diseases and were matched with each scleroderma patient for sex and biopsy site (five men and five women; aged 27-49 years). Primary cultures were established in DMEM media with 10% fetal calf serum (FCS), 2 mM L-glutamine, 50 pg/ml gentamicin and amphotericin (first culture only, 5 pg/ml). These monolayer cultures were maintained at 37°C in 90% air and 10% CO2. Fibroblasts between the third and sixth passage were studied, which were almost all components of these cultures after these passages. In some cases, fibroblasts from same skin biopsies were examined in all three, four, five and six passages. Nuclear run on transcription assay Nuclei were prepared from the cells by lysing the cells in the solution which contained 10 mm Tris (pH 7 5), 2 mm MgCl2, 3
Correspondence: Y. Deguchi, MD, Ph.D, National Institute of Health, 10687 Weymouth Street no. 103, Bethesda, MD 20814, USA.
97
Y. Deguchi, N. Shibata & S. Kishimoto
98
Table 1. Examined skin fibroblasts from patients with scleroderma
pg). The final washes were done with 0-2 x SSC at 450C for I h, before autoradiography (Piguet et al., 1989).
Fibroblast no. Sex Age (years) Area of biopsy
Statistical analysis Statistical analysis of the data was performed using Student's ttest.
2 3 4
5 6 7 8 9 10 11 12 13 14 15
M M M M M M M F F F F F F F F
32 29 40 42 33 36 30 29 35 33 40 47 31 39 34
forearm forearm forearm forearm pretibial pretibial pretibial forearm forearm forearm forearm pretibial pretibial pretibial pretibial
mM CaC12, 5 mm DTT and 0-02% of NP40, with subsequent centrifugation through 2 M sucrose solution. Three million nuclei were suspended into 100 p1 of 50% glycerol solution with 50 mm Tris (pH 7-5), 5 mm MgCl2 and 0 1 mm EDTA. The suspension of nuclei was immediately mixed with an equal volume of buffer containing 0-2 M KC1, 5 mM MgCl2, 5 mM DTT, 1 mm of ATP, CTP, GTP, and 200 U of RNAsin (RNAse inhibitor, 500 units, Amersham International, Amersham, UK). The preparation was then incubated at 28°C for 20 min after addition of 50 pCi 32P-radiolabelled UTP (3000 mCi/ml, Amersham). To the preparation SDS and EDTA solution were added to a final concentration of 1% and 5 mm, respectively, followed by treatment with proteinase K (I mg/ml) at 42°C for 30 min. RNA was extracted with phenol and chloroform from the preparation and precipitated with ethanol. The pellet was resuspended into 3 ml of hybridization buffer which contained 50% of formamide, 0-75 M NaCl, 0-5% SDS, 2 mm EDTA, 50 mM HEPES (pH 7 0), 1/10 dilution of Denhardt's solution and denatured salmon sperm DNA (500 pg/ml) (Marzluff& Huang, 1984). Finally, the preparation was applied to the nitrocellulose filter onto which the human hsp 70 cDNA probe (1 -2 kb, BamHI fragment, a kind gift from Dr H. Ariga, Japan) (Reed et al., 1987), or beta-actin probe (Wako Pure Chemical Industries, Japan) had been dotted. After 24-h incubation, the filter was washed three times in 0-2 x SSC and 0 I% SDS at 45°C, dried and exposed to X-ray film with intensifying screen at - 70°C. In some experiments, the hybridized dot was excised from the filter and directly counted by a beta-counter (Deguchi, Negoro & Kishimoto, 1988). RNA preparation and Northern blot assay Total RNA from the cells was prepared by the guanidiniumthiocyanate and caesium chloride procedure, denatured in glyoxal, and electrophoresed in 1 4% agarose gels (20 pg/lane) (Deguchi et al., 1988). RNA was transferred to nylon membranes (Biodyne). The filters were hybridized (Thomas, 1980) in a solution including nick-translated 32P-labelled human hsp 70 cDNA probe (1 -2 kb, BamHI fragment) or actin probe (Wako Pure Chemical Industries) (specific activity 1-5 x 108 ct/min per
RESULTS The transcriptional level of some genes, such as c-fos and c-myc protooncogenes (Kelly et al., 1983), is very low in quiescent cells and changes after stimulation with serum or growth factors. Fibroblasts can be made quiescent either by growth to confluency in the presence of serum or by removal of it. In the present study, we focused on quantitative comparison of hsp 70 gene expression in scleroderma and normal fibroblasts. As shown in Fig. I (nuclear run on transcription assay), the transcriptional level of hsp 70 gene was always high in scleroderma fibroblasts at the quiescent condition in the absence of serum. Equal numbers of nuclei (I12 x 107) from each experimental condition were used for this assay. As evaluated by beta-counter, scleroderma cells showed 8-6-fold increases in the transcriptional level of hsp 70 gene expression in the absence of serum, in comparison to normal controls (Fig. 2). We used three to six passages of cultured fibroblasts. As show in Table 2, within these passages the differences were not affected by cell passage. After stimulation with serum, hsp 70 gene is activated N3
N7
S3
S7
Fig. 1. Nuclear run on transcription assay of heat shock protein 70 gene in scleroderma (S) and normal (N) fibroblasts. Equal numbers (1-5 x 106) of nuclei from cells starved for 48 h in 1% FCS (1) and cells induced for 12 h with 10% FCS (2) were tested.
SA NA
SQ NQ 0
8
16
24
hsp 70 transcription (relative counts 105 cells) Fig. 2. Quantification of heat shock protein (hsp) 70 gene transcription in scleroderma (S) and normal (N) fibroblasts with serum activation (A) and at quiescent condition (Q). The relative amount of hsp 70 gene transcription present (relative counts/105 cells) was determined using a beta-counter to measure hybridized filters of nuclear run on transcription assay. Mean value+s.d. of all samples (15 scleroderma and 10 controls) is presented.
99
Heat shock protein gene in scleroderma Table 2. Contribution of the number of cell passages to hsp 70 transcription
a
N3
S15 2
2
Hsp 70 transcription* (relative counts/105 cells) Cultured with Fibroblast Number of 10% FCS no. cell passages 1% FCS 2 2 2 2
3 4 5 6
16 19 17 20
21 20 28 22
5 5 5 5
3 4 5 6
23 16 20
23 25 29 29
24
* The values were determined by beta-counter of nuclear run on transcription assay of hsp 70 gene, as shown in the legend of Fig. 3.
N3
N7
S3
S7
2
Fig. 3. Nuclear run on transcription assay of beta-actin gene in scleroderma (S) and normal (N) fibroblasts. Equal numbers (5 x 106) of nuclei from cells starved for 48 h in 1% FCS (1) and cells induced for 12 h with 10% FCS (2) were tested.
and the transcriptional levels of hsp 70 gene were almost similar in scleroderma and normal fibroblasts (Figs 1, 2). We found no significant changes in transcriptional level of beta-actin gene expression in scleroderma and normal fibroblasts after serum deprivation or after stimulation with serum (Fig. 3). Abnormal gene expression is often caused by genomic DNA alterations, such as gene amplification and chromosomal translocations. We checked the possible alterations of the hsp 70 gene in scleroderma fibroblasts by Southern blot assay. Neither gene amplification nor detectable gene translocations were seen (data not shown). We further studied total RNA from scleroderma fibroblasts by Northern blot assay. As shown in Fig. 4, we also found hsp 70 message of normal size in scleroderma fibroblasts. The increased levels of amounts of hsp 70 transcripts at serum starvation in scleroderma versus control fibroblasts obtained by counting the Northern blots (7-5-fold) were similar to the results obtained by nuclear run on transcription assay (8 6-fold). It is well known that the half life of hsp 70 transcripts is very short (Linquist & Craig, 1988). The Northern blots in this study can reflect the transcriptional level of hsp 70 gene. These results suggest that the posttranscriptional changes of hsp 70 transcripts are not mainly affected in scleroderma fibroblasts and that increased amount of hsp 70 transcripts in scleroderma fibroblasts is partly due to activated transcription of the gene.
b
Fig. 4. Heat shock protein (hsp) 70 gene transcripts in scleroderma (S) and normal (N) fibroblasts. Northern blot analysis of total RNA (10 mg) from cells starved for 48 h in 1% FCS (1) and subsequently induced for 12 h with 10% FCS (2) was performed as described in Materials and Methods. (a) hsp 70 probe; (b) beta-actin probe.
DISCUSSION Scleroderma is a disease characterized by diffuse sclerosis, often affecting skin, lung and kidney. We have demonstrated here that the transcriptional level of hsp 70 gene is increased in scleroderma fibroblasts in comparison to normal controls, and is markedly different at quiescent condition in the absence of serum. We have further shown that the induction of hsp 70 gene by adding serum is not significant in scleroderma fibroblasts. In eukaryotes, the transcription of hsp gene has been investigated intensively in Drosophila (Lindquist, 1980). The activation of hsp gene transcription is mediated by specific sequence elements in the heat shock promoters. Deletion analyses revealed that 20 bp located about 20 nucleotides upstream of 5'-portion to the TATA box were essential for induction of hsp 70 gene in Drosophila (Parker & Topol, 1984). The sequences were recognized by multi-meric DNA binding proteins (Parker & Topol, 1984). We found no significant gene alterations in sclerotic fibroblasts DNA by Southern blot assay. It is not clear yet how the spontaneous elevation of transcription of hsp 70 gene in scleroderma fibroblasts is regulated by the similar DNAbinding proteins. It is well known that scleroderma fibroblasts show no evidence of transformation. Recently it has been suggested that hsp could play an essential role in cell growth and differentiation (Lindquist & Craig, 1988). Some growth factors and mitogens increase hsp 70 transcripts in mammalian fibroblasts and lymphocytes (Granelli-Piperno, Andrus & Steinman, 1986). Taken together, our findings suggest that all or some populations of scleroderma fibroblasts are activated and that intracellular events, including gene expression, change in scleroderma fibroblasts. The reduction in difference in hsp expression between scleroderma and normal fibroblasts by culturing in the presence compared with the absence of serum allows a preliminary analysis of possible factors, such as cytokines which could be
Y. Deguchi, N. Shibata & S. Kishimoto
100
added to the system. In our preliminary experiments we observed that skin fibroblasts show increased expression of hsp 70 gene after stimulation by interleukin-I and transforming growth factor-beta (TGF-f) (submitted for publication). It could be that intracellular events activated by cytokine networks in fibroblasts are altered and/or dysregulated in scleroderma fibroblasts. We therefore propose that an understanding of intracellular events in scleroderma fibroblasts, involved in hsp gene activation, and of the role of hsp products especially can give some new approaches to pathogenesis for chronic inflammation of scleroderma fibrosis. The study of the mechanisms and clinical meaning of spontaneous activation of hsp gene in scleroderma fibroblasts is in progress.
ACKNOWLEDGMENTS We thank Dr H. Ariga for the generous gift of the human hsp 70 cDNA probe and Dr T. Ohkubo and Dr J. Curran for valuable discussions. This study was supported in part by grants from the Ministry of Culture and Education and Science of Japan.
REFERENCES BARNETT, A.J. (1978) Scleroderma (progressive systemic sclerosis): progress and course based on a personal series of 118 cases. Med. J. Aust. 2, 129. DEGUCHI, Y., NEGORO, S. & KISHIMOTO, S. (1988) Age-related changes of heat shock protein gene transcription in human peripheral blood mononuclear cells. Biochem. Biophys. Res. Comm. 157, 580.
GRANELLI-PIPERNO, A., ANDRUS, L. & STEINMAN, R.M. (1986) Lymphokine and non-lymphokine mRNA levels in stimulated human T cells. J. exp. Med. 163, 922. HAYNES, D.C. & GERSHWIN, M.E. (1982) Immunopathology of progressive systemic sclerosis (PSS). Semin. Arthritis Rheum. 11, 331. KELLY, K., COCHRANN, B., STILES, C.D. & LEDER, P. (1983) Activation of protooncogenes in mitogen stimulated lymphocytes. Cell, 35, 603. LEROY, E.C., BLACK, C., FLEISCHMAJER, R., JABLONSKA, S., KRIEG, T., MEDSGER, T.A., ROWELL, N. & WOLLHEIM, F. (1988) Scleroderma: classification, subsets and pathogenesis. J. Rheumatol. 15, 202. LINQUIST, S. (1980) Varying pattern of protein synthesis in drosophila during heat shock: implications for regulation. Dev. Biol. 77, 463. LINDQUIST, S. & CRAIG, E.A. (1988) The heat-shock proteins. Annu. Rev. Genet. 22, 631. MARZLUFF, W.F. & HUANG, R.C.C. (1984) Nuclear run on transcription assay. In Transcription and Translation (ed. by B.D. Hames & S.J. Higgins), p. 89. I.R.L. Press, Oxford. MORANGE, M., DIN, A., BENSAUDE, 0. & BABINET, C. (1984) Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells. Mol. Cell Biol. 4, 730. PARKER, C.S. & TOPOL, J. (1984) A drosophila RNA polymerase II transcription factor binds to the regulatory site of an hsp 70 gene. Cell, 37, 273. PIGUET, P.F., COLLART, M.A., GARU, G.E., KAPANCI, Y. & VASSALI, P. (1989) Tumor necrosis factor/cachectin plays a key role in bleomycininduced pneumopathy and fibroblasts. J. exp. Med. 170, 655. REED, J.C., TsuJIMOTO, Y., ALPERS, J.D., CROCE, C.M. & NOWELL, P.C. (1987) Regulation of bc1-2 protooncogene expression during normal human lymphocyte proliferation. Science, 237, 1295. SCHLESINGER, M.J., ALIPERTI, G. & KELLY, P.M. (1982) The response of cells to heat shock. Trends Biochem. Sci. 7, 222. THOMAS, P.S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl Acad. Sci. USA, 77, 5201. TROJANOWSKA, M., Wu, L. & LEROY, E.C. (1988) Elevated expression of c-myc protooncogene in scleroderma. Oncogene, 3, 477.
Elevated transcription of heat shock protein gene in scleroderma fibroblasts Y. DEGUCHI, N. SHIBATA* & S. KISHIMOTO Third Department of Internal Medicine, Osaka University School of Medicine, Osaka and * Department of Internal Medicine, Centre for Adult Diseases in Osaka, Osaka, Japan (Acceptedfor publication 22 January 1990)
SUMMARY Scleroderma is a systemic disorder characterized by fibrosis, which affects skin, lung, kidney and other organs. Heat shock protein (hsp) (70 kD) has been implicated as an essential element of cell function in cell growth and differentiation. To study the molecular basis of intracellular events in scleroderma fibroblasts, we compared the expression of hsp 70 gene in scleroderma and normal control fibroblasts by nuclear run on transcription assay and Northern blot assay. We show that scleroderma fibroblasts express more than eight times higher level of hsp 70 transcription in normal control fibroblasts at quiescent conditions in the absence of serum. After stimulation with serum, the transcription level of the hsp 70 gene is similar in scleroderma and normal control fibroblasts. Therefore, our results indicate an alteration/activation of intracellular events in scleroderma fibroblasts.
Keywords heat shock protein
gene
transcription scleroderma fibrosis
INTRODUCTION
In order to study the molecular basis of intracellular disorders in scleroderma fibroblasts, we examined hsp 70 gene expression in scleroderma and control fibroblasts.
In systemic autoimmune disorders such as scleroderma, connective tissue inflammation and microvascular damage resulting in multi-organ system involvement are the most prominent pathological changes. Scleroderma is a systemic disorder characterized by fibrosis and over-production of extracellular matrix components (Haynes & Gershwin, 1982). The mechanisms of fibrosis in scleroderma is not clearly known. Recently it was suggested that some population of fibroblasts in fibrotic lesion is changed and causes over-production of extracellular matrix (LeRoy et al., 1988; Trojanowska, Wu & LeRoy, 1988). Most of the intracellular disorders in sclerotic fibroblasts are still unknown. Changes of gene expression in response to heat shock stress have been described in human cells (Lindquist & Craig, 1988). The general effect of heat shock stress suppresses normal protein synthesis and enhances the synthesis of proteins such as heat shock proteins (hsp). Schlesinger, Aliperti & Kelly (1982) showed that various chemical, mechanical and environmental stresses could induce hsp; and the expression of hsp was regulated developmentally and in cell differentiation (Morange et al., 1984). It has been suggested that these proteins could be important for essential functions in cell growth and differentiation (Morange et al., 1984). The hsp of the 70 kD (hsp 70) family includes the inducible and cognate 68 and 72 kD hsp. Hsp 70 is the most conserved and well characterized.
MATERIALS AND METHODS
Skin biopsies and cell culture Skin fibroblasts were obtained by skin biopsy from affected portions of 15 scleroderma patients with diffuse cutaneous systemic sclerosis, as shown in Table 1. The diagnosis of scleroderma was made clinically in addition to the biopsy findings characterized by thinned epidermis with loss of rete pegs and a marked increase in collagen fibres within the dermis (Barnett, 1978). Control fibroblasts were obtained by forearm or pretibial skin biopsy of healthy donors who had no history of rheumatic diseases and were matched with each scleroderma patient for sex and biopsy site (five men and five women; aged 27-49 years). Primary cultures were established in DMEM media with 10% fetal calf serum (FCS), 2 mM L-glutamine, 50 pg/ml gentamicin and amphotericin (first culture only, 5 pg/ml). These monolayer cultures were maintained at 37°C in 90% air and 10% CO2. Fibroblasts between the third and sixth passage were studied, which were almost all components of these cultures after these passages. In some cases, fibroblasts from same skin biopsies were examined in all three, four, five and six passages. Nuclear run on transcription assay Nuclei were prepared from the cells by lysing the cells in the solution which contained 10 mm Tris (pH 7 5), 2 mm MgCl2, 3
Correspondence: Y. Deguchi, MD, Ph.D, National Institute of Health, 10687 Weymouth Street no. 103, Bethesda, MD 20814, USA.
97
Y. Deguchi, N. Shibata & S. Kishimoto
98
Table 1. Examined skin fibroblasts from patients with scleroderma
pg). The final washes were done with 0-2 x SSC at 450C for I h, before autoradiography (Piguet et al., 1989).
Fibroblast no. Sex Age (years) Area of biopsy
Statistical analysis Statistical analysis of the data was performed using Student's ttest.
2 3 4
5 6 7 8 9 10 11 12 13 14 15
M M M M M M M F F F F F F F F
32 29 40 42 33 36 30 29 35 33 40 47 31 39 34
forearm forearm forearm forearm pretibial pretibial pretibial forearm forearm forearm forearm pretibial pretibial pretibial pretibial
mM CaC12, 5 mm DTT and 0-02% of NP40, with subsequent centrifugation through 2 M sucrose solution. Three million nuclei were suspended into 100 p1 of 50% glycerol solution with 50 mm Tris (pH 7-5), 5 mm MgCl2 and 0 1 mm EDTA. The suspension of nuclei was immediately mixed with an equal volume of buffer containing 0-2 M KC1, 5 mM MgCl2, 5 mM DTT, 1 mm of ATP, CTP, GTP, and 200 U of RNAsin (RNAse inhibitor, 500 units, Amersham International, Amersham, UK). The preparation was then incubated at 28°C for 20 min after addition of 50 pCi 32P-radiolabelled UTP (3000 mCi/ml, Amersham). To the preparation SDS and EDTA solution were added to a final concentration of 1% and 5 mm, respectively, followed by treatment with proteinase K (I mg/ml) at 42°C for 30 min. RNA was extracted with phenol and chloroform from the preparation and precipitated with ethanol. The pellet was resuspended into 3 ml of hybridization buffer which contained 50% of formamide, 0-75 M NaCl, 0-5% SDS, 2 mm EDTA, 50 mM HEPES (pH 7 0), 1/10 dilution of Denhardt's solution and denatured salmon sperm DNA (500 pg/ml) (Marzluff& Huang, 1984). Finally, the preparation was applied to the nitrocellulose filter onto which the human hsp 70 cDNA probe (1 -2 kb, BamHI fragment, a kind gift from Dr H. Ariga, Japan) (Reed et al., 1987), or beta-actin probe (Wako Pure Chemical Industries, Japan) had been dotted. After 24-h incubation, the filter was washed three times in 0-2 x SSC and 0 I% SDS at 45°C, dried and exposed to X-ray film with intensifying screen at - 70°C. In some experiments, the hybridized dot was excised from the filter and directly counted by a beta-counter (Deguchi, Negoro & Kishimoto, 1988). RNA preparation and Northern blot assay Total RNA from the cells was prepared by the guanidiniumthiocyanate and caesium chloride procedure, denatured in glyoxal, and electrophoresed in 1 4% agarose gels (20 pg/lane) (Deguchi et al., 1988). RNA was transferred to nylon membranes (Biodyne). The filters were hybridized (Thomas, 1980) in a solution including nick-translated 32P-labelled human hsp 70 cDNA probe (1 -2 kb, BamHI fragment) or actin probe (Wako Pure Chemical Industries) (specific activity 1-5 x 108 ct/min per
RESULTS The transcriptional level of some genes, such as c-fos and c-myc protooncogenes (Kelly et al., 1983), is very low in quiescent cells and changes after stimulation with serum or growth factors. Fibroblasts can be made quiescent either by growth to confluency in the presence of serum or by removal of it. In the present study, we focused on quantitative comparison of hsp 70 gene expression in scleroderma and normal fibroblasts. As shown in Fig. I (nuclear run on transcription assay), the transcriptional level of hsp 70 gene was always high in scleroderma fibroblasts at the quiescent condition in the absence of serum. Equal numbers of nuclei (I12 x 107) from each experimental condition were used for this assay. As evaluated by beta-counter, scleroderma cells showed 8-6-fold increases in the transcriptional level of hsp 70 gene expression in the absence of serum, in comparison to normal controls (Fig. 2). We used three to six passages of cultured fibroblasts. As show in Table 2, within these passages the differences were not affected by cell passage. After stimulation with serum, hsp 70 gene is activated N3
N7
S3
S7
Fig. 1. Nuclear run on transcription assay of heat shock protein 70 gene in scleroderma (S) and normal (N) fibroblasts. Equal numbers (1-5 x 106) of nuclei from cells starved for 48 h in 1% FCS (1) and cells induced for 12 h with 10% FCS (2) were tested.
SA NA
SQ NQ 0
8
16
24
hsp 70 transcription (relative counts 105 cells) Fig. 2. Quantification of heat shock protein (hsp) 70 gene transcription in scleroderma (S) and normal (N) fibroblasts with serum activation (A) and at quiescent condition (Q). The relative amount of hsp 70 gene transcription present (relative counts/105 cells) was determined using a beta-counter to measure hybridized filters of nuclear run on transcription assay. Mean value+s.d. of all samples (15 scleroderma and 10 controls) is presented.
99
Heat shock protein gene in scleroderma Table 2. Contribution of the number of cell passages to hsp 70 transcription
a
N3
S15 2
2
Hsp 70 transcription* (relative counts/105 cells) Cultured with Fibroblast Number of 10% FCS no. cell passages 1% FCS 2 2 2 2
3 4 5 6
16 19 17 20
21 20 28 22
5 5 5 5
3 4 5 6
23 16 20
23 25 29 29
24
* The values were determined by beta-counter of nuclear run on transcription assay of hsp 70 gene, as shown in the legend of Fig. 3.
N3
N7
S3
S7
2
Fig. 3. Nuclear run on transcription assay of beta-actin gene in scleroderma (S) and normal (N) fibroblasts. Equal numbers (5 x 106) of nuclei from cells starved for 48 h in 1% FCS (1) and cells induced for 12 h with 10% FCS (2) were tested.
and the transcriptional levels of hsp 70 gene were almost similar in scleroderma and normal fibroblasts (Figs 1, 2). We found no significant changes in transcriptional level of beta-actin gene expression in scleroderma and normal fibroblasts after serum deprivation or after stimulation with serum (Fig. 3). Abnormal gene expression is often caused by genomic DNA alterations, such as gene amplification and chromosomal translocations. We checked the possible alterations of the hsp 70 gene in scleroderma fibroblasts by Southern blot assay. Neither gene amplification nor detectable gene translocations were seen (data not shown). We further studied total RNA from scleroderma fibroblasts by Northern blot assay. As shown in Fig. 4, we also found hsp 70 message of normal size in scleroderma fibroblasts. The increased levels of amounts of hsp 70 transcripts at serum starvation in scleroderma versus control fibroblasts obtained by counting the Northern blots (7-5-fold) were similar to the results obtained by nuclear run on transcription assay (8 6-fold). It is well known that the half life of hsp 70 transcripts is very short (Linquist & Craig, 1988). The Northern blots in this study can reflect the transcriptional level of hsp 70 gene. These results suggest that the posttranscriptional changes of hsp 70 transcripts are not mainly affected in scleroderma fibroblasts and that increased amount of hsp 70 transcripts in scleroderma fibroblasts is partly due to activated transcription of the gene.
b
Fig. 4. Heat shock protein (hsp) 70 gene transcripts in scleroderma (S) and normal (N) fibroblasts. Northern blot analysis of total RNA (10 mg) from cells starved for 48 h in 1% FCS (1) and subsequently induced for 12 h with 10% FCS (2) was performed as described in Materials and Methods. (a) hsp 70 probe; (b) beta-actin probe.
DISCUSSION Scleroderma is a disease characterized by diffuse sclerosis, often affecting skin, lung and kidney. We have demonstrated here that the transcriptional level of hsp 70 gene is increased in scleroderma fibroblasts in comparison to normal controls, and is markedly different at quiescent condition in the absence of serum. We have further shown that the induction of hsp 70 gene by adding serum is not significant in scleroderma fibroblasts. In eukaryotes, the transcription of hsp gene has been investigated intensively in Drosophila (Lindquist, 1980). The activation of hsp gene transcription is mediated by specific sequence elements in the heat shock promoters. Deletion analyses revealed that 20 bp located about 20 nucleotides upstream of 5'-portion to the TATA box were essential for induction of hsp 70 gene in Drosophila (Parker & Topol, 1984). The sequences were recognized by multi-meric DNA binding proteins (Parker & Topol, 1984). We found no significant gene alterations in sclerotic fibroblasts DNA by Southern blot assay. It is not clear yet how the spontaneous elevation of transcription of hsp 70 gene in scleroderma fibroblasts is regulated by the similar DNAbinding proteins. It is well known that scleroderma fibroblasts show no evidence of transformation. Recently it has been suggested that hsp could play an essential role in cell growth and differentiation (Lindquist & Craig, 1988). Some growth factors and mitogens increase hsp 70 transcripts in mammalian fibroblasts and lymphocytes (Granelli-Piperno, Andrus & Steinman, 1986). Taken together, our findings suggest that all or some populations of scleroderma fibroblasts are activated and that intracellular events, including gene expression, change in scleroderma fibroblasts. The reduction in difference in hsp expression between scleroderma and normal fibroblasts by culturing in the presence compared with the absence of serum allows a preliminary analysis of possible factors, such as cytokines which could be
Y. Deguchi, N. Shibata & S. Kishimoto
100
added to the system. In our preliminary experiments we observed that skin fibroblasts show increased expression of hsp 70 gene after stimulation by interleukin-I and transforming growth factor-beta (TGF-f) (submitted for publication). It could be that intracellular events activated by cytokine networks in fibroblasts are altered and/or dysregulated in scleroderma fibroblasts. We therefore propose that an understanding of intracellular events in scleroderma fibroblasts, involved in hsp gene activation, and of the role of hsp products especially can give some new approaches to pathogenesis for chronic inflammation of scleroderma fibrosis. The study of the mechanisms and clinical meaning of spontaneous activation of hsp gene in scleroderma fibroblasts is in progress.
ACKNOWLEDGMENTS We thank Dr H. Ariga for the generous gift of the human hsp 70 cDNA probe and Dr T. Ohkubo and Dr J. Curran for valuable discussions. This study was supported in part by grants from the Ministry of Culture and Education and Science of Japan.
REFERENCES BARNETT, A.J. (1978) Scleroderma (progressive systemic sclerosis): progress and course based on a personal series of 118 cases. Med. J. Aust. 2, 129. DEGUCHI, Y., NEGORO, S. & KISHIMOTO, S. (1988) Age-related changes of heat shock protein gene transcription in human peripheral blood mononuclear cells. Biochem. Biophys. Res. Comm. 157, 580.
GRANELLI-PIPERNO, A., ANDRUS, L. & STEINMAN, R.M. (1986) Lymphokine and non-lymphokine mRNA levels in stimulated human T cells. J. exp. Med. 163, 922. HAYNES, D.C. & GERSHWIN, M.E. (1982) Immunopathology of progressive systemic sclerosis (PSS). Semin. Arthritis Rheum. 11, 331. KELLY, K., COCHRANN, B., STILES, C.D. & LEDER, P. (1983) Activation of protooncogenes in mitogen stimulated lymphocytes. Cell, 35, 603. LEROY, E.C., BLACK, C., FLEISCHMAJER, R., JABLONSKA, S., KRIEG, T., MEDSGER, T.A., ROWELL, N. & WOLLHEIM, F. (1988) Scleroderma: classification, subsets and pathogenesis. J. Rheumatol. 15, 202. LINQUIST, S. (1980) Varying pattern of protein synthesis in drosophila during heat shock: implications for regulation. Dev. Biol. 77, 463. LINDQUIST, S. & CRAIG, E.A. (1988) The heat-shock proteins. Annu. Rev. Genet. 22, 631. MARZLUFF, W.F. & HUANG, R.C.C. (1984) Nuclear run on transcription assay. In Transcription and Translation (ed. by B.D. Hames & S.J. Higgins), p. 89. I.R.L. Press, Oxford. MORANGE, M., DIN, A., BENSAUDE, 0. & BABINET, C. (1984) Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells. Mol. Cell Biol. 4, 730. PARKER, C.S. & TOPOL, J. (1984) A drosophila RNA polymerase II transcription factor binds to the regulatory site of an hsp 70 gene. Cell, 37, 273. PIGUET, P.F., COLLART, M.A., GARU, G.E., KAPANCI, Y. & VASSALI, P. (1989) Tumor necrosis factor/cachectin plays a key role in bleomycininduced pneumopathy and fibroblasts. J. exp. Med. 170, 655. REED, J.C., TsuJIMOTO, Y., ALPERS, J.D., CROCE, C.M. & NOWELL, P.C. (1987) Regulation of bc1-2 protooncogene expression during normal human lymphocyte proliferation. Science, 237, 1295. SCHLESINGER, M.J., ALIPERTI, G. & KELLY, P.M. (1982) The response of cells to heat shock. Trends Biochem. Sci. 7, 222. THOMAS, P.S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl Acad. Sci. USA, 77, 5201. TROJANOWSKA, M., Wu, L. & LEROY, E.C. (1988) Elevated expression of c-myc protooncogene in scleroderma. Oncogene, 3, 477.
