ORIGINAL
RESEARCH
Hepatocyte Growth Factor and p38 Promote Osteogenic Differentiation of Human Mesenchymal Stem Cells Kristina K. Aenlle, Kevin M. Curtis, Bernard A. Roos, and Guy A. Howard Geriatric Research, Education, and Clinical Center and Research Service (K.K.A., K.M.C., B.A.R., G.A.H.), Veterans Affairs Medical Center, Miami, Florida 33125; Departments of Medicine (B.A.R., G.A.H.), Neurology (B.A.R.), and Biochemistry & Molecular Biology (K.M.C., G.A.H.), University of Miami Miller School of Medicine, Miami, Florida 33101
Hepatocyte growth factor (HGF) is a paracrine factor involved in organogenesis, tissue repair, and wound healing. We report here that HGF promotes osteogenic differentiation through the transcription of key osteogenic markers, including osteocalcin, osterix, and osteoprotegerin in human mesenchymal stem cells and is a necessary component for the establishment of osteoblast mineralization. Blocking endogenous HGF using PHA665752, a c-Met inhibitor (the HGF receptor), or an HGF-neutralizing antibody attenuates mineralization, and PHA665752 markedly reduced alkaline phosphatase activity. Moreover, we report that HGF promotion of osteogenic differentiation involves the rapid phosphorylation of p38 and differential regulation of its isoforms, p38␣ and p38. Western blot analysis revealed a significantly increased level of p38␣ and p38 protein, and reverse transcription quantitative PCR revealed that HGF increased the transcriptional level of both p38␣ and p38. Using small interfering RNA to reduce the transcription of p38␣ and p38, we saw differential roles for p38␣ and p38 on the HGF-induced expression of key osteogenic markers. In summary, our data demonstrate the importance of p38 signaling in HGF regulation of osteogenic differentiation. (Molecular Endocrinology 28: 722–730, 2014)
epatocyte growth factor (HGF), also known as scatter factor, is a pleiotropic factor that regulates cell proliferation, motility, morphogenesis, and angiogenesis. HGF has also been shown to participate in organogenesis, tissue repair, neuronal induction, and bone remodeling (1). HGF acts as a paracrine factor throughout the body by binding to its tyrosine kinase receptor, c-Met, and activating a signal transduction cascade. HGF has robust effects on human mesenchymal stem cells (hMSCs); blocking HGF bioactivity reduces hMSC migration (2) and blocking the HGF receptor (c-MET) diminishes both proliferation and differentiation (3). hMSCs are precursors for the generation of different mesenchymal cell lineages, including osteoblasts, adipocytes, myoblasts, chondroblasts, and fibroblasts (3). MSCs have been used to repair bone defects in animal models (4 – 8) and MSCs
H
overexpressing bone morphogenetic protein-2 were shown to restore bone defects in aged rats (9). Along these lines, HGF likely primes hMSCs toward the osteoblastic pathway. Therefore, HGF as a driver of hMSC osteogenic differentiation has the potential to promote bone repair and maintain bone homeostasis, but information on the mechanism(s) of HGF-induced hMSC-mediated bone repair/regeneration is lacking. HGF activates an array of signaling cascades in hMSCs, including rapid phosphorylation of ERK, AKT/PI3K (10, 11), and as we report here, p38. p38 MAPK consists of 4 identified isoforms, p38␣, p38, p38␦, and p38␥, and the rapid induction of p38␣ and p38 isoforms has been reported to be important for skeletogenesis and bone homeostasis (12). Moreover, the loss of p38 signaling through the knockout of p38 phosphatases, MKK3 and
ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received September 13, 2013. Accepted March 15, 2014. First Published Online March 27, 2014
Abbreviations: BSP2, bone sialoprotein 2; COL1a1, collagen 1a1; FBS, fetal bovine serum; HGF, hepatocyte growth factor; hMSCs, human mesenchymal stem cells; OCN, osteocalcin; OPG, osteoprotegerin, OSX, osterix; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA.
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MKK6, and the loss and/or reduction of p38␣ and - lead to a decrease in bone maturation and maintenance (12). p38 has been shown to directly phosphorylate RUNX2 (leading to possible protein stabilization) and is involved in promoting RUNX2 and osterix (OSX) gene expression (13, 14), key transcription factors for osteoblast differentiation. p38 has also been reported to be involved in bone morphogenetic protein-4 gene expression, which is important for bone formation and osteoblast differentiation (15, 16). Taken together, the data presented here highlight the important role that p38 plays on bone formation and maintenance and osteoblast differentiation. The current study was done to determine the role of HGF/c-MET, through the activation of p38, during hMSC osteogenic differentiation and whether blocking p38␣ or p38 inhibited HGF-induced expression of key osteoblast markers. The results of this study, showing that HGF promotes osteogenic maturation of hMSCs through the p38 pathway, provide important insight into the role of HGF during osteogenic differentiation and led to a better understanding of the possible mechanism of HGF/p38 signaling in the therapeutic potential of hMSCs.
Materials and Methods Bone marrow-derived mesenchymal stem cell (MSCs) isolation and cell culture hMSCs were isolated and cultured as previously described (17), from postmortem thoracolumbar (T1–L5) vertebral bodies of donors of various ages (4 –30 years of age) immediately after death from traumatic injury. hMSCs from a 4- and 7-yearold male were used in these studies. Guidelines were followed as outlined by the Committee on the Use of Human Subjects in Research at the University of Miami. Cells were grown in Expansion media, DMEM-low glucose media, containing 10% fetal bovine serum (FBS, Hyclone Laboratories; catalog no. 30039), 20 mM ascorbic acid (Fluka/Sigma; catalog no. 49752), an essential fatty acid solution (18), and antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin) (Gibco: catalog no. 15140) at 37°C on 10 ng/mL fibronectin (Sigma; catalog no. F2518)coated flasks (Nunclon) in 21% O2, 5% CO2, and 92% N2. Media was changed every 3 days, and cells were subcultured upon reaching 80% confluence. In expansion conditions cells were plated at 1000 cells/cm2.
Osteogenic differentiation During differentiation, cells were grown in Osteogenic media, ␣MEM-low glucose media, containing 10% FBS (Hyclone; lot no. 30039), 20 mM ascorbic acid (Fluka/Sigma; catalog no. 49752), an essential fatty acid solution (18), antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin) (Gibco; catalog no. 15140), and 10 mM -glycerophosphate at 37°C on 10 ng/mL fibronectin (Sigma; catalog no. F2518)-coated flasks (Nunclon) in 21% O2, 5% CO2, and 92% N2. Cells for osteogenic differ-
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entiation were plated at 10 000 cells/cm2 , and day 0 began when cells were 100% confluent with subsequent media changes every 3– 4 days.
HGF treatment Human MSCs were cultured in T75 flasks in standard growth medium (10% FBS). At 50% confluence, cells were treated with 10, 20, 40, or 100 ng/mL HGF (Peprotech) dissolved in 0.01% BSA in PBS. The ideal concentration of HGF was determined to be 40 ng/mL and was used for the subsequent experiments. For long-term experiments (ie, mineralization) HGF was added during media changes, which occurred every 3– 4 days. For short-term experiments a single treatment of HGF was used.
Cell proliferation assay Cells were plated at 1000 cells/cm2, and on the following day (day 0) they were treated with or without HGF neutralizing antibody MAB294 at either 100 ng/mL or 300 ng/mL. After 30 minutes, cells were treated with or without HGF (40 ng/mL). Cells were allowed to proliferate for 5 days. On day 5, cells were counted using a hemocytometer.
Western blot Whole-cell lysates were prepared with Nonidet P-40 lysis buffer. Protein concentration was determined using BCA system (Thermo Fisher Scientific). Western immunoblotting was performed as described previously (19). Briefly, 5 g of total protein was resolved on 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Millipore Corp). The membranes were blocked with 5% nonfat milk in PBS buffer containing 0.1% Tween 20 for 1 hour, and then incubated at 4°C overnight with primary antibodies against phospho-p38, p38, p38␣, p38 (each at 1:1000; Cell Signaling Technology), phospho-p38␣ (Millipore,) and ␣-tubulin (1:1000; Santa Cruz Biotechnology). After overnight incubation unbound primary antibodies were removed by rinsing the membranes 3 ⫻ for 10 minutes with Tris-buffered saline. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000) for 1 hour at room temperature and then rinsed 3 ⫻ for 10 minutes with Tris-buffered saline. ECL-plus Western Blot system (Amersham Bioscience) was used to image the protein bands. Band intensities were compared using ImageJ (NIH, Bethesda, MA) and densitometric analysis was obtained by normalizing to loading control (␣-tubulin) and where appropriate to total p38 or total cMet.
RNA interference with p38 and p38 isoforms Transient transfection of small interfering RNA (siRNA) in hMSCs was performed using TransIT-TKO transfection reagent with either p38␣ siRNA, p38 siRNA, or nonsilencing control siRNA (a non homologous, scrambled sequence equivalent) (Cell Signaling Technology). In brief, hMSCs were harvested by trypsinization and plated at 3000 cells/cm2 in 6-well plates. Within 24 hours, 250 L of Opti-Mem I-reduced Serum Media, 10 l of TransIT-TKO transfection reagent, and 100 nM control siRNA or p38 siRNA were combined and incubated at room temperature for 30 minutes. Following incubation TKO/ siRNA complex was added drop-wise to wells containing cells. hMSCs were transfected for 4 days; at that time protein was
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formamide [Sigma] and 24 mg of fast blue BB [Sigma] in 30 mL 100 mM TrisHCL, pH 9.6, and 10 mg of MgCl2 (Sigma) in final mixed stain solution with pH 9.0). Cells were rinsed extensively with water, and images were taken by inverted microscope with camera.
Mineralization assay hMSCs were seeded in 12-well plates (100 000 cells per well). The next day media were replaced with osteogenic media and cells were treated with Figure 1. HGF increases the level of key osteogenic markers. RT-qPCR of OCN, Osterix, OPG, c-MET inhibitor PHA665752 (Santa Collagen1a1, and Bone Sialoprotein2 (BSP2) at 1, 2, and 3 days after a single treatment of HGF Cruz Biotechnology) at concentrations (40 ng/mL). Relative values were calculated by ⌬⌬CT method, with each gene normalized to of 0 (C), 0.05, 0.1, 0.25, 0.5, or 1 M at housekeeping gene RPL13␣ and its control (day 0). Error bars represent SD of triplicate experiments, with *, P ⬍ .05 vs control (Student’s t test). Control has no error bar because the each media change for 17 days. The values obtained for each experiment were set at 1 for subsequent fold change calculation. same protocol was used with HGF neutralizing antibody, at concentrations of 5, 10, 100, 200, and 300 ng/mL collected to determine knockdown level of p38␣ and p38. At (MAB294; R&D systems) for a total of 10 days. Cells were fixed the beginning of day 4 the cells were treated with HGF (40 with 2% paraformaldehyde /0.2% gluteraldehyde for 1 hour, ng/mL). RNA from cells treated with p38␣ siRNA, p38 and then stained with 40 mM alizarin red-S as previously desiRNA, and control siRNA was collected after 2 days of subsescribed (17). Stain was quantified (562 nM) after solubilizing in quent HGF treatment. 10% cetylpyridinium chloride (10 mM sodium phosphate, pH 7.0) (20). Reverse transcription-quantitative PCR (RT-qPCR) Total RNA was isolated from hMSCs with RNAqueous4PCR Kit following manufacturer’s procedures (Ambion). The amount and quality of total RNA were determined using a Nanodrop spectrophotometer. cDNA was created by reverse transcribing 2 g of total RNA with SuperScript II (Invitrogen) following manufacturer’s instructions. For RT-qPCR, 100 ng of cDNA was added to 12.5 L of SYBRGreen, 0.375 L ROX, 3.125 L H2O, and 2 L primer pairs. PCR was amplified using a Stratagene 3500 set at 40 cycles of 94°C for 1 minute, 55 to 60°C for 1 minute, and 72°C for 2 minutes. The ⌬⌬CT method, using RPL13a as the internal control, was used to analyze the results.
ELISA The level of endogenous HGF was determined from cell culture supernates. Briefly, cellular media were collected, centrifruged at 1000 ⫻ g for 15 minutes to remove any cellular debris, and supernatant was then collected and stored frozen (⫺80°C) until the ELISA was performed. ELISA was performed according to manufacturer’s protocol (R&D Systems). Each sample was run in duplicate, with 3 separate experiments.
Alkaline phosphatase activity hMSCs were seeded in 12-well plates (100 000 cells per well). The next day media were replaced with osteogenic media, and cells were treated with c-MET inhibitor PHA665752 (Santa Cruz Biotechnology) at concentrations of 0 (C), 0.05, 0.1, 0.25, 0.5, or 1 M for 3 days. Alkaline phosphatase activity was evaluated at the end of culture day 3. hMSC alkaline phosphatase activity was measured in triplicate as previously described (17). Briefly, cells were rinsed with PBS, fixed with ice-cold 2% paraformaldehyde/0.2% gluteraldehyde for 1 hour at 4°C, and then stained for 30 minutes at 37°C with stain solution (8 mg naphthol AS-TR phosphate [Sigma] in 0.3 mL N-N⬘-dimethyl-
Statistical analysis Experiments were performed in triplicate. Group data are represented as mean ⫾ SD. Quantitative data were analyzed using Student’s t test; a value of P ⬍ .05 was considered significant.
Results HGF increases the expression of key osteogenic markers To determine the role of HGF on hMSC osteogenic differentiation/maturation, we used RT-qPCR to evaluate the effect of HGF treatment on the gene expression of key osteogenic markers, osteocalcin (OCN), OSX, osteoprotegerin (OPG), bone sialoprotein 2 (BSP2), and collagen 1a1 (COL1a1) (Figure 1). One day after a single treatment with HGF (40 ng/mL) revealed significantly (P ⬍ .05) increased expression of OCN. By day 2 (48 hours after treatment) all 5 genes examined displayed a significant increase in gene expression. At day 3 (72 hours after HGF treatment) the gene expression level of OCN remained significantly greater than the basal (control) levels, whereas OSX, OPG, COL1a1, and BSP2 returned to basal levels. Using the ⌬⌬CT method our results show that after 2 days of HGF treatment OCN displayed the largest increase in expression with an 11-fold increase over basal levels, whereas OSX displayed a roughly 6-fold increase in gene expression compared with basal levels.
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and the rate of HGF production/utilization was maintained in a steady state through day 17.
Figure 2. Endogenous level of HGF in expansion and differentiation conditions. Endogenous levels of HGF in cell media were determined by ELISA at 550 nM. A, Media were collected from expansion conditions on day 0 (control), 1, 2, 3, and 6. B, Media were collected from differentiation conditions on day 0 (control), 3, 7, 10, 14, and 17. The average absorbance was normalized to total cell number. *, P ⬍ .05 vs control using Student’s t test; #, P ⬍ .05 vs day 1 using Student’s t test.
OPG displayed a roughly 3-fold increase in gene expression 2 days after HGF treatment. To determine the level of endogenous HGF during expansion of hMSCs and subsequent differentiation, we used ELISA. HGF rapidly increased in the culture media within 24 hours of media changes in expansion conditions (Figure 2A). In expansion conditions cells were plated at 1000 cells/cm2 (ie, cells were only 30%– 40% confluent at the start of the experiment). The level of HGF quickly decreases after 2 days in expansion media with significant decreases from days 1– 6 (P ⬍ .05). In contrast, the level of HGF remained in a steady state during differentiation (Figure 2B). In differentiation conditions, cells were plated at 10 000 cells/cm2, and experiments began when cells were 100% confluent. The level of HGF quickly accumulated after 3 days in differentiation media,
Blocking endogenous HGF signaling disrupts mineralization To further characterize the role of HGF on osteogenic maturation, we blocked the HGF signaling pathway by 2 independent methods. In the first, HGF-neutralizing antibody significantly reduced mineralization in a dose-dependent manner. As expected in these cells, there was very little mineralization at day 7, and no significant differences were seen with or without HGF-neutralizing antibody at day 7 (Figure 3, A and B). However, at day 10, a significant dose response was detected in mineralization using HGF-neutralizing antibody. Although 5 and 10 ng/mL showed no significant change in mineralization compared with control, 100, 200, and 300 ng/mL of HGF-neutralizing antibody caused significant decreases in mineralization (P ⬍ .05) (Figure 3, A and B). To show that HGF-neutralizing antibody is blocking HGF activity we performed a cell proliferation assay. At day 5, cell proliferation was significantly (P ⬍ .05) lower in HGFtreated cells, as measured by total cell number. HGFneutralizing antibody alone had no significant effect on cell proliferation compared with control. Most importantly, HGF-neutralizing antibody blocked the HGF-induced decrease in cell proliferation. Cells treated with 100 ng/mL of HGF-neutralizing antibody plus HGF (40 ng/ mL) did not significantly differ from control cells but did proliferate significantly more than HGF-treated cells (P ⬍ .05). A higher dose of neutralizing antibody (300 ng/mL) in combination with HGF (40 ng/mL) also displayed a significantly higher cell proliferation than HGF-treated cells (P ⬍ .05) but also was significantly lower in cell proliferation than control (P ⬍ .05). To show that the effect of HGF on mineralization was through its receptor, c-MET, we treated hMSCs with PHA665752, a c-MET inhibitor, and then quantified calcium accumulation during mineralization. We observed a significant (P ⬍ .05), concentration-dependent (0.1–1 M) decrease in mineralization across days 10 and 17 compared with control (Figure 4, A and B). PHA665752 at a lower dose, 0.05 M, was able to significantly reduce mineralization at day 10 but did not maintain the attenuated mineralization through day 17. Blocking cMET activation with PHA665752 also caused a significant, dose-dependent decrease in alkaline phosphatase activity. Three days after PHA665752 treatment (0.1, 0.25, 0.5, and 1 M) hMSCs displayed reduced alkaline phosphatase activity (Figure 4 C). PHA665752 at a lower dose, 0.05 M, did not attenuate alkaline phosphatase activity 3 days after treatment.
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utes. Phosphorylation of p38 was not determined at this time due to the lack of a specific phospho-p38 antibody. HFG increases the level of p38␣ and p38 To determine whether HGF treatment on p38 signal transduction is isoform specific, we performed Western blot and RT-qPCR analyses on p38␣ and p38 isoforms. A single treatment of HGF (40 ng/mL) led to a transient increase in p38␣ and p38 in hMSCs. Western blot analysis showed an increase in both p38␣ and p38 on day 1. Both p38␣ and p38 maintained protein expression through days 2 and 3, with Figure 3. HGF neutralizing antibody reduces mineralization. hMSCs were treated with 0 (C), 5, p38 displaying the highest increase 10, 100, 200, or 300 ng/mL of HGF neutralizing antibody (R&D; MAB294) during media changes in protein expression compared every 3– 4 days and then stained using Alizarin Red-S on days 7 and 10 (panel A). Mineralization with control (Figure 6A). RT-qPCR was then quantified and absorbance measured at 550 nm (panel B). Cell proliferation assay of analysis of p38 gene expression after HGF neutralizing antibody (100 and 300 ng/mL) with or without HGF (40 ng/mL) at day 5 (panel C). Error bars represent SD. *, P ⬍ .05 vs control by Student’s t test; #, P ⬍ .05 vs HGF-treated a single HGF treatment revealed difusing Student’s t test. C, control. ferential expression of p38␣ and p38 (Figure 6B). HGF (40 ng/mL) did not significantly affect gene exWestern blot analysis showed that at doses of 0.1 and 1.0 M, PHA665752 did block HGF-induced activation of pression levels of p38␣ after 1 day of treatment. However, cMET (ie, phospho-cMET) (Figure 4D). Densitometric 2 days of HGF treatment did show a significant increase analysis showed that HGF alone produced an increase of in the gene expression of p38␣ (P ⬍ .05), followed by a 3.5 (350% increase) compared with control. Doses of 0.1 significant decrease (P ⬍ 0,05) in the gene expression of and 1.0 M of PHA665752 produced cMet activation of p38␣ at day 3. HGF significantly altered the level of 1.9 and 1.2 (PHA665752 ⫹HGF), roughly 50% and p38. On day 1 expression level of p38 gene was signif70% decreases in activation compared with HGF treated, icantly increased by HGF treatment (P ⬍ .05). The most robust response (P ⬍ .0001) to HGF treatment was seen respectively. at day 2, with a 10-fold increase in expression compared with basal levels of p38 expression. The gene expression HGF rapidly phosphorylates p38 in a dose- and time-dependent manner level of p38 returned to basal levels by day 3. In view of the reports cited above that p38 is involved in skeletogenesis, we performed a dose response and time p38␣ and p38 differentially regulate HGF-induced course of HGF treatment to determine whether HGF af- expression of key osteogenic markers siRNA was used to determine the role(s) of p38␣ and fects p38 signal transduction in hMSCs. HGF (40 ng/mL)  in HGF induction of osteogenic markers. Two days p38 treatment of hMSCs showed a significant increase in phosphorylation of total p38 and continued with 100 of HGF treatment in siControl-treated cells significantly ng/mL after 10 minutes of treatment (Figure 5A). HGF (P ⬍ .05) increased the transcription level of OPG and concentrations of 10 and 20 ng/mL did not significantly OSX compared with siControl, with a 5.5- and 1.5-fold increase p38 phosphorylation within 10 minutes of treat- (respectively) increase. A significant increase was not seen ment. A time course with 40 ng/mL HGF led to significant for OCN (P ⫽ .06), COL1a1, or BSP2 when comparing phosphorylation of p38␣ within 5 minutes and phosphor- siControl vs siControl ⫹HGF, although a significant inylation of total p38 within 10 minutes (Figure 5B). Phos- crease was seen in Figure 1. Compared with the results phorylation of both p38␣ and total p38 peaked at 10 –15 seen in Figure 1, Figure 7 displays the results after a total minutes and subsequently decreased at 30 and 60 min- of 6 days in culture. The change in HGF levels between
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receiving HGF. However, the increase in OPG was truncated compared with siControl and p38␣ siRNA treated with HGF; they displayed a roughly 5.5- and 3.7-fold, respectively, increase.
Discussion The data presented herein is the first account describing the interaction of HGF, p38␣, and p38 on osteogenic maturation. The HGF/p38 signal transduction pathway provides a novel mode of regulation/support for bone maintenance and bone renewal. HGF plays a vital role in bone maintenance and bone renewal through the activation of key osteoFigure 4. cMET inhibitor, PHA665752, blocks mineralization. hMSCs were treated with 0 (C), genic markers and promoting osteo0.05, 0.1, 0.25, 0.5, or 1 M cMET inhibitor (PHA665752) during media changes every 3– 4 days blast proliferation (1, 21). In paraland then stained using Alizarin Red-S on days 10 and 17 (panel A). Mineralization was then quantified and absorbance measured at 550 nM (panel B). Alkaline phophosphatase activity was lel, our data show that HGF induces determined at day 3 (panel C). Western blot analysis showed a reduction of phosphorylated cMet the expression of OCN, OPG, and total cMet (␣-tubulin as a loading control), with densitometry quantification shown (panel COL1a1, BSP2, and OSX in a tranD). Error bars represent SD. *, P ⬍ .05 vs control using Student’s t test. C, control. sient manner. Although gene expression of these key osteogenic markers day 2 and day 6 may be contributing to the lack of effect begins to rise in hMSC after 1 day of HGF treatment, we of HGF on siControl. When HGF is added to the MSCs on day 2, the level of HGF is still elevated and/or the rate observed the most robust effect at day 2, with the levels of turnover is lower than on day 6. It is possible that the then returning to baseline by day 3. Similar to our data, 40 ng/mL plus the elevated level of endogenous HGF is HGF (30 ng/mL) has also been reported to induce osteowhat is needed to drive the gene transcription of OCN pontin gene expression within 24 hours (21). Using a and other osteogenic markers. Also it is possible that on rabbit model of osteonecrosis, Wen et al (22) reported an day 6 the rate of turnover is too high for the 40 ng/mL of improvement in tissue repair when transplanted MSCs were transfected with HGF. In their model, HGF levels HGF to have an effect on siControl-treated cells. The results of HGF treatment in cells treated with ei- immediately increased following transplantation and did ther p38␣ siRNA or p38 siRNA show significant not decline until 2 weeks after transplantation. Recent changes in gene expression. RT-qPCR data reveal a sig- work by Tomson et al (23) showed an increase in OSX nificant (P ⬍ .05) increase in the expression of OCN, and OCN gene expression after 7 and 12 days of treatOPG, and COL1a1 when HGF-treated cells were exposed ment with HGF. In those studies experimental media to p38␣ siRNA. However, although the data show a were changed every 48 hours, suggesting a cumulative trend, HGF did not significantly increase the expression effect of HGF treatment after continued treatment (23). of OSX or BSP2 in p38␣ siRNA-exposed cells. Most im- Along these lines, we have previously reported that HGF portantly, HGF did not induce the expression of OCN, inhibits cell proliferation and, combined with 1␣,25-diOSX, or COL1a1 in cells in which the level of p38 was hydroxy vitamin D, promotes osteogenic differentiation decreased. Cells treated with p38 siRNA failed to re- (3, 18, 31). Thus it appears that both acute and chronic spond to HGF induction of gene expression, displaying a treatment with HGF promotes osteogenesis. Using chemical inhibition of the HGF pathway we saw similar level of transcriptional activity as cells treated with p38 siRNA without HGF. OPG and BSP2 did show a marked reduction in mineralization and alkaline phosa significant increase in gene expression in p38 siRNA phatase activity. hMSCs produce HGF, and endogenous HGF-treated cells compared with p38 siRNA cells not HGF can have autocrine and paracrine activity on the
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The role of p38 in bone development and bone maintenance is becoming widely recognized. p38␣-knockout mice have reduced bone mass, and mice lacking p38␣ in osteoblasts display an age-dependent decrease in bone mass and bone mineral density (12, 24). Similarly, p38knockout mice also exhibit a diminished bone mass (12). Greenblatt et al (12) hypothesized that p38␣ and p38 have different functions in bone development and maintenance: p38␣ regulating early osteoblast differentiation and p38 regulating late osteoblast maturation. Our data show that p38 appears to have a major role in the HGF induction of genes associated with mature osteoblasts (eg, OCN). For example, HGF induction of OCN and Col1a1 are dependent on p38. OCN, secreted solely by osteoblast, is necessary for alkaline phosphatase activity and mineralization. Additionally, HGF was not able to induce Figure 5. HGF activation of p38 is dose and time dependent. Dose OXS gene expression in p38␣- or p38 siRNA-treated response (panel A) of HGF treatment (10 minutes) of hMSCs. Time course cells. Therefore, OSX, a key mediator of early osteoblast (panel B) of HGF treatment (40 ng/mL) of hMSCs. Western blot analyses were performed on phosphorylated-p38␣ (p-p38␣), total p38␣ (p38␣), differentiation, appears to require both p38␣ and p38 phosphorylated-p38 (p-p38), and total p38 (p38), with ␣-tubulin as a for full HGF induction. The HGF-induced expression of loading control (n ⫽ 3 independent experiments in triplicate, with a OPG was truncated, but not removed, with either p38␣representative blot and densitometric quantification shown). or p38 siRNA. In contrast, BSP2 showed significant incells (3). Blocking the endogenous HGF activity, through crease in gene expression in p38 siRNA-treated cells but the inhibition of c-MET activity or with an antibody neu- not in p38␣ siRNA-treated cells. Hence, our data support tralizing endogenous HGF, was sufficient to attenuate the role of HGF and p38 in early and late stages of osteoosteogenic differentiation of hMSCs. These results sug- blast differentiation. gest that basal HGF activity is promoting bone mainteAlthough p38␣ is the most abundant p38 isoform (as nance/osteoblast differentiation. Although we did not see seen in our hMSCs, data not shown), the role of p38 is an increase in mineralization or alkaline phosphatase important for bone mass. As indicated above, p38(data not shown) using 40 ng/mL of HGF, it is likely due knockout mice display a reduction in bone mass (12). to a ceiling effect with the endogenous HGF (Figure 2B) Interestingly, p38␣ is the major isoform found in osteactivity combined with the differentiation media rapidly oclasts and is essential for osteoclast differentiation and pushing the cells toward maturation/mineralization and bone resorption (25). Our data suggest that p38 is necincreasing alkaline phosphatase activity, thus making it essary for the induction of gene expression of osteogenic difficult to see an increase in our system. markers, but it is unclear whether this is due to the loss of p38-mediated gene expression or whether p38␣ is acting as a direct inhibitor of genes involved in osteogenic differentiation. Moreover, the rapid activation of p38 by HGF also contributes to osteogenic differentiation. Here we show that HGF rapidly activates p38 via phosphorylation within 10 minutes of treatment. This effect is dose dependent, with at Figure 6. HGF increases the level of p38 protein (panel A) and mRNA expression (panel B). least 40 ng/mL of HGF needed to see Western blot analysis (panel A) of p38␣ and p38 after a single treatment with HGF (40 ng/mL) a significant increase in p38 phoson days 0, 1, 2, and 3. (n ⫽ 3 independent experiments in triplicate with a representative blot phorylation. HGF treatment also and densitometric quantification shown). B, RT-qPCR of p38 isoforms after HGF treatment (40 ng/mL) on days 0, 1, 2, and 3. Relative values were calculated by ⌬⌬CT method, with each gene leads to increases in p38␣ and p38 normalized to housekeeping gene RPL13␣ and its control (day 0). Error bars represent SD of protein levels and in their gene extriplicate experiments; *, P ⬍ .05 vs control; and **, P ⬍ .0001 vs control using Student’s t test. pression. It is likely that HGF is inControl has no error bar because the values obtained for each experiment were set at 1 for fluencing the expression of p38 subsequent fold change calculation.
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Figure 7. p38 and p38␣ mediate HGF-induced osteogenic gene expression. RT-qPCR was performed on RNA from hMSCs exposed to 4 days of siRNA control, siRNA p38␣, or siRNA p38. On day 4 the cells were then treated or not with HGF (40 ng/mL) for an additional 2 days (6 days total). Relative values were calculated by ⌬⌬CT method, with each gene normalized to housekeeping gene RPL13␣ and its control (day 0) (left panels and 2 lower right panels). Western blot analysis of the knockdown of p38␣ and p38 4 days after siRNA treatment (upper right panel). Error bars represent SD of 3 independent experiments; *, P ⬍ .05 vs control using Student’s t test. siControl has no error bar because the values obtained for each experiment were set at 1 for subsequent fold change calculation.
through the rapid phosphorylation of p38, and thereby maintaining the protein levels, ie, activated p38 is less likely to be degraded. Additionally, the increase in gene expression can lead to increased protein levels by days 2 and 3. HGF’s dual induction and maintenance of p38 activity appear to have numerous effects on hMSCs, including the induction of key osteogenic proteins. The p38 MAPK pathway is likely promoting osteoblast differentiation and maturation by acting as a mediator of both the activation of key osteogenic proteins and transcriptional activity of key osteogenic genes. Phosphorylation of p38 leads to downstream effects including the activation and expression of RUNX2 and OSX (26 – 29). Moreover, p38 plays a key role in OSX-induced expression of COL1a1 (30). Finally, p38 has been reported to be essential in mineralization and alkaline phosphatase activity during osteogenesis in MC3T3-E1 cells (31, 32), in which p38 promotes the transcription of osteogenic genes through posttranscriptional modification.
The role of p38 in HGF-mediated osteogenic differentiation and bone repair has promising therapeutic potential in that promotion of p38 over p38␣ might lead to a stronger HGF effect in osteogenic maturation and bone repair. The combination of p38 activity and HGF treatment in hMSCs may have important clinical applications for the treatment of bone diseases and the promotion of bone health.
Acknowledgments We thank David Vazquez and B. Nubia Rodriguez for excellent technical assistance. Address all correspondence to: Guy A. Howard, Veterans Affairs Medical Center GRECC (11GRC), 1201 NW 16th Street, Miami, FL 33125. E-mail: [email protected]. For reprints contact: [email protected]. This work was supported by Merit Review awards from the Department of Veterans Affairs (to B.A.R., G.A.H.) and the
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National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award 7F32AR062990 (to K.M.C.). Dr Howard is the recipient of a Senior Research Career Scientist award from the Department of Veterans Affairs. Disclosure Summary: The authors have nothing to disclose.
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RESEARCH
Hepatocyte Growth Factor and p38 Promote Osteogenic Differentiation of Human Mesenchymal Stem Cells Kristina K. Aenlle, Kevin M. Curtis, Bernard A. Roos, and Guy A. Howard Geriatric Research, Education, and Clinical Center and Research Service (K.K.A., K.M.C., B.A.R., G.A.H.), Veterans Affairs Medical Center, Miami, Florida 33125; Departments of Medicine (B.A.R., G.A.H.), Neurology (B.A.R.), and Biochemistry & Molecular Biology (K.M.C., G.A.H.), University of Miami Miller School of Medicine, Miami, Florida 33101
Hepatocyte growth factor (HGF) is a paracrine factor involved in organogenesis, tissue repair, and wound healing. We report here that HGF promotes osteogenic differentiation through the transcription of key osteogenic markers, including osteocalcin, osterix, and osteoprotegerin in human mesenchymal stem cells and is a necessary component for the establishment of osteoblast mineralization. Blocking endogenous HGF using PHA665752, a c-Met inhibitor (the HGF receptor), or an HGF-neutralizing antibody attenuates mineralization, and PHA665752 markedly reduced alkaline phosphatase activity. Moreover, we report that HGF promotion of osteogenic differentiation involves the rapid phosphorylation of p38 and differential regulation of its isoforms, p38␣ and p38. Western blot analysis revealed a significantly increased level of p38␣ and p38 protein, and reverse transcription quantitative PCR revealed that HGF increased the transcriptional level of both p38␣ and p38. Using small interfering RNA to reduce the transcription of p38␣ and p38, we saw differential roles for p38␣ and p38 on the HGF-induced expression of key osteogenic markers. In summary, our data demonstrate the importance of p38 signaling in HGF regulation of osteogenic differentiation. (Molecular Endocrinology 28: 722–730, 2014)
epatocyte growth factor (HGF), also known as scatter factor, is a pleiotropic factor that regulates cell proliferation, motility, morphogenesis, and angiogenesis. HGF has also been shown to participate in organogenesis, tissue repair, neuronal induction, and bone remodeling (1). HGF acts as a paracrine factor throughout the body by binding to its tyrosine kinase receptor, c-Met, and activating a signal transduction cascade. HGF has robust effects on human mesenchymal stem cells (hMSCs); blocking HGF bioactivity reduces hMSC migration (2) and blocking the HGF receptor (c-MET) diminishes both proliferation and differentiation (3). hMSCs are precursors for the generation of different mesenchymal cell lineages, including osteoblasts, adipocytes, myoblasts, chondroblasts, and fibroblasts (3). MSCs have been used to repair bone defects in animal models (4 – 8) and MSCs
H
overexpressing bone morphogenetic protein-2 were shown to restore bone defects in aged rats (9). Along these lines, HGF likely primes hMSCs toward the osteoblastic pathway. Therefore, HGF as a driver of hMSC osteogenic differentiation has the potential to promote bone repair and maintain bone homeostasis, but information on the mechanism(s) of HGF-induced hMSC-mediated bone repair/regeneration is lacking. HGF activates an array of signaling cascades in hMSCs, including rapid phosphorylation of ERK, AKT/PI3K (10, 11), and as we report here, p38. p38 MAPK consists of 4 identified isoforms, p38␣, p38, p38␦, and p38␥, and the rapid induction of p38␣ and p38 isoforms has been reported to be important for skeletogenesis and bone homeostasis (12). Moreover, the loss of p38 signaling through the knockout of p38 phosphatases, MKK3 and
ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received September 13, 2013. Accepted March 15, 2014. First Published Online March 27, 2014
Abbreviations: BSP2, bone sialoprotein 2; COL1a1, collagen 1a1; FBS, fetal bovine serum; HGF, hepatocyte growth factor; hMSCs, human mesenchymal stem cells; OCN, osteocalcin; OPG, osteoprotegerin, OSX, osterix; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA.
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MKK6, and the loss and/or reduction of p38␣ and - lead to a decrease in bone maturation and maintenance (12). p38 has been shown to directly phosphorylate RUNX2 (leading to possible protein stabilization) and is involved in promoting RUNX2 and osterix (OSX) gene expression (13, 14), key transcription factors for osteoblast differentiation. p38 has also been reported to be involved in bone morphogenetic protein-4 gene expression, which is important for bone formation and osteoblast differentiation (15, 16). Taken together, the data presented here highlight the important role that p38 plays on bone formation and maintenance and osteoblast differentiation. The current study was done to determine the role of HGF/c-MET, through the activation of p38, during hMSC osteogenic differentiation and whether blocking p38␣ or p38 inhibited HGF-induced expression of key osteoblast markers. The results of this study, showing that HGF promotes osteogenic maturation of hMSCs through the p38 pathway, provide important insight into the role of HGF during osteogenic differentiation and led to a better understanding of the possible mechanism of HGF/p38 signaling in the therapeutic potential of hMSCs.
Materials and Methods Bone marrow-derived mesenchymal stem cell (MSCs) isolation and cell culture hMSCs were isolated and cultured as previously described (17), from postmortem thoracolumbar (T1–L5) vertebral bodies of donors of various ages (4 –30 years of age) immediately after death from traumatic injury. hMSCs from a 4- and 7-yearold male were used in these studies. Guidelines were followed as outlined by the Committee on the Use of Human Subjects in Research at the University of Miami. Cells were grown in Expansion media, DMEM-low glucose media, containing 10% fetal bovine serum (FBS, Hyclone Laboratories; catalog no. 30039), 20 mM ascorbic acid (Fluka/Sigma; catalog no. 49752), an essential fatty acid solution (18), and antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin) (Gibco: catalog no. 15140) at 37°C on 10 ng/mL fibronectin (Sigma; catalog no. F2518)coated flasks (Nunclon) in 21% O2, 5% CO2, and 92% N2. Media was changed every 3 days, and cells were subcultured upon reaching 80% confluence. In expansion conditions cells were plated at 1000 cells/cm2.
Osteogenic differentiation During differentiation, cells were grown in Osteogenic media, ␣MEM-low glucose media, containing 10% FBS (Hyclone; lot no. 30039), 20 mM ascorbic acid (Fluka/Sigma; catalog no. 49752), an essential fatty acid solution (18), antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin) (Gibco; catalog no. 15140), and 10 mM -glycerophosphate at 37°C on 10 ng/mL fibronectin (Sigma; catalog no. F2518)-coated flasks (Nunclon) in 21% O2, 5% CO2, and 92% N2. Cells for osteogenic differ-
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entiation were plated at 10 000 cells/cm2 , and day 0 began when cells were 100% confluent with subsequent media changes every 3– 4 days.
HGF treatment Human MSCs were cultured in T75 flasks in standard growth medium (10% FBS). At 50% confluence, cells were treated with 10, 20, 40, or 100 ng/mL HGF (Peprotech) dissolved in 0.01% BSA in PBS. The ideal concentration of HGF was determined to be 40 ng/mL and was used for the subsequent experiments. For long-term experiments (ie, mineralization) HGF was added during media changes, which occurred every 3– 4 days. For short-term experiments a single treatment of HGF was used.
Cell proliferation assay Cells were plated at 1000 cells/cm2, and on the following day (day 0) they were treated with or without HGF neutralizing antibody MAB294 at either 100 ng/mL or 300 ng/mL. After 30 minutes, cells were treated with or without HGF (40 ng/mL). Cells were allowed to proliferate for 5 days. On day 5, cells were counted using a hemocytometer.
Western blot Whole-cell lysates were prepared with Nonidet P-40 lysis buffer. Protein concentration was determined using BCA system (Thermo Fisher Scientific). Western immunoblotting was performed as described previously (19). Briefly, 5 g of total protein was resolved on 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Millipore Corp). The membranes were blocked with 5% nonfat milk in PBS buffer containing 0.1% Tween 20 for 1 hour, and then incubated at 4°C overnight with primary antibodies against phospho-p38, p38, p38␣, p38 (each at 1:1000; Cell Signaling Technology), phospho-p38␣ (Millipore,) and ␣-tubulin (1:1000; Santa Cruz Biotechnology). After overnight incubation unbound primary antibodies were removed by rinsing the membranes 3 ⫻ for 10 minutes with Tris-buffered saline. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000) for 1 hour at room temperature and then rinsed 3 ⫻ for 10 minutes with Tris-buffered saline. ECL-plus Western Blot system (Amersham Bioscience) was used to image the protein bands. Band intensities were compared using ImageJ (NIH, Bethesda, MA) and densitometric analysis was obtained by normalizing to loading control (␣-tubulin) and where appropriate to total p38 or total cMet.
RNA interference with p38 and p38 isoforms Transient transfection of small interfering RNA (siRNA) in hMSCs was performed using TransIT-TKO transfection reagent with either p38␣ siRNA, p38 siRNA, or nonsilencing control siRNA (a non homologous, scrambled sequence equivalent) (Cell Signaling Technology). In brief, hMSCs were harvested by trypsinization and plated at 3000 cells/cm2 in 6-well plates. Within 24 hours, 250 L of Opti-Mem I-reduced Serum Media, 10 l of TransIT-TKO transfection reagent, and 100 nM control siRNA or p38 siRNA were combined and incubated at room temperature for 30 minutes. Following incubation TKO/ siRNA complex was added drop-wise to wells containing cells. hMSCs were transfected for 4 days; at that time protein was
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formamide [Sigma] and 24 mg of fast blue BB [Sigma] in 30 mL 100 mM TrisHCL, pH 9.6, and 10 mg of MgCl2 (Sigma) in final mixed stain solution with pH 9.0). Cells were rinsed extensively with water, and images were taken by inverted microscope with camera.
Mineralization assay hMSCs were seeded in 12-well plates (100 000 cells per well). The next day media were replaced with osteogenic media and cells were treated with Figure 1. HGF increases the level of key osteogenic markers. RT-qPCR of OCN, Osterix, OPG, c-MET inhibitor PHA665752 (Santa Collagen1a1, and Bone Sialoprotein2 (BSP2) at 1, 2, and 3 days after a single treatment of HGF Cruz Biotechnology) at concentrations (40 ng/mL). Relative values were calculated by ⌬⌬CT method, with each gene normalized to of 0 (C), 0.05, 0.1, 0.25, 0.5, or 1 M at housekeeping gene RPL13␣ and its control (day 0). Error bars represent SD of triplicate experiments, with *, P ⬍ .05 vs control (Student’s t test). Control has no error bar because the each media change for 17 days. The values obtained for each experiment were set at 1 for subsequent fold change calculation. same protocol was used with HGF neutralizing antibody, at concentrations of 5, 10, 100, 200, and 300 ng/mL collected to determine knockdown level of p38␣ and p38. At (MAB294; R&D systems) for a total of 10 days. Cells were fixed the beginning of day 4 the cells were treated with HGF (40 with 2% paraformaldehyde /0.2% gluteraldehyde for 1 hour, ng/mL). RNA from cells treated with p38␣ siRNA, p38 and then stained with 40 mM alizarin red-S as previously desiRNA, and control siRNA was collected after 2 days of subsescribed (17). Stain was quantified (562 nM) after solubilizing in quent HGF treatment. 10% cetylpyridinium chloride (10 mM sodium phosphate, pH 7.0) (20). Reverse transcription-quantitative PCR (RT-qPCR) Total RNA was isolated from hMSCs with RNAqueous4PCR Kit following manufacturer’s procedures (Ambion). The amount and quality of total RNA were determined using a Nanodrop spectrophotometer. cDNA was created by reverse transcribing 2 g of total RNA with SuperScript II (Invitrogen) following manufacturer’s instructions. For RT-qPCR, 100 ng of cDNA was added to 12.5 L of SYBRGreen, 0.375 L ROX, 3.125 L H2O, and 2 L primer pairs. PCR was amplified using a Stratagene 3500 set at 40 cycles of 94°C for 1 minute, 55 to 60°C for 1 minute, and 72°C for 2 minutes. The ⌬⌬CT method, using RPL13a as the internal control, was used to analyze the results.
ELISA The level of endogenous HGF was determined from cell culture supernates. Briefly, cellular media were collected, centrifruged at 1000 ⫻ g for 15 minutes to remove any cellular debris, and supernatant was then collected and stored frozen (⫺80°C) until the ELISA was performed. ELISA was performed according to manufacturer’s protocol (R&D Systems). Each sample was run in duplicate, with 3 separate experiments.
Alkaline phosphatase activity hMSCs were seeded in 12-well plates (100 000 cells per well). The next day media were replaced with osteogenic media, and cells were treated with c-MET inhibitor PHA665752 (Santa Cruz Biotechnology) at concentrations of 0 (C), 0.05, 0.1, 0.25, 0.5, or 1 M for 3 days. Alkaline phosphatase activity was evaluated at the end of culture day 3. hMSC alkaline phosphatase activity was measured in triplicate as previously described (17). Briefly, cells were rinsed with PBS, fixed with ice-cold 2% paraformaldehyde/0.2% gluteraldehyde for 1 hour at 4°C, and then stained for 30 minutes at 37°C with stain solution (8 mg naphthol AS-TR phosphate [Sigma] in 0.3 mL N-N⬘-dimethyl-
Statistical analysis Experiments were performed in triplicate. Group data are represented as mean ⫾ SD. Quantitative data were analyzed using Student’s t test; a value of P ⬍ .05 was considered significant.
Results HGF increases the expression of key osteogenic markers To determine the role of HGF on hMSC osteogenic differentiation/maturation, we used RT-qPCR to evaluate the effect of HGF treatment on the gene expression of key osteogenic markers, osteocalcin (OCN), OSX, osteoprotegerin (OPG), bone sialoprotein 2 (BSP2), and collagen 1a1 (COL1a1) (Figure 1). One day after a single treatment with HGF (40 ng/mL) revealed significantly (P ⬍ .05) increased expression of OCN. By day 2 (48 hours after treatment) all 5 genes examined displayed a significant increase in gene expression. At day 3 (72 hours after HGF treatment) the gene expression level of OCN remained significantly greater than the basal (control) levels, whereas OSX, OPG, COL1a1, and BSP2 returned to basal levels. Using the ⌬⌬CT method our results show that after 2 days of HGF treatment OCN displayed the largest increase in expression with an 11-fold increase over basal levels, whereas OSX displayed a roughly 6-fold increase in gene expression compared with basal levels.
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and the rate of HGF production/utilization was maintained in a steady state through day 17.
Figure 2. Endogenous level of HGF in expansion and differentiation conditions. Endogenous levels of HGF in cell media were determined by ELISA at 550 nM. A, Media were collected from expansion conditions on day 0 (control), 1, 2, 3, and 6. B, Media were collected from differentiation conditions on day 0 (control), 3, 7, 10, 14, and 17. The average absorbance was normalized to total cell number. *, P ⬍ .05 vs control using Student’s t test; #, P ⬍ .05 vs day 1 using Student’s t test.
OPG displayed a roughly 3-fold increase in gene expression 2 days after HGF treatment. To determine the level of endogenous HGF during expansion of hMSCs and subsequent differentiation, we used ELISA. HGF rapidly increased in the culture media within 24 hours of media changes in expansion conditions (Figure 2A). In expansion conditions cells were plated at 1000 cells/cm2 (ie, cells were only 30%– 40% confluent at the start of the experiment). The level of HGF quickly decreases after 2 days in expansion media with significant decreases from days 1– 6 (P ⬍ .05). In contrast, the level of HGF remained in a steady state during differentiation (Figure 2B). In differentiation conditions, cells were plated at 10 000 cells/cm2, and experiments began when cells were 100% confluent. The level of HGF quickly accumulated after 3 days in differentiation media,
Blocking endogenous HGF signaling disrupts mineralization To further characterize the role of HGF on osteogenic maturation, we blocked the HGF signaling pathway by 2 independent methods. In the first, HGF-neutralizing antibody significantly reduced mineralization in a dose-dependent manner. As expected in these cells, there was very little mineralization at day 7, and no significant differences were seen with or without HGF-neutralizing antibody at day 7 (Figure 3, A and B). However, at day 10, a significant dose response was detected in mineralization using HGF-neutralizing antibody. Although 5 and 10 ng/mL showed no significant change in mineralization compared with control, 100, 200, and 300 ng/mL of HGF-neutralizing antibody caused significant decreases in mineralization (P ⬍ .05) (Figure 3, A and B). To show that HGF-neutralizing antibody is blocking HGF activity we performed a cell proliferation assay. At day 5, cell proliferation was significantly (P ⬍ .05) lower in HGFtreated cells, as measured by total cell number. HGFneutralizing antibody alone had no significant effect on cell proliferation compared with control. Most importantly, HGF-neutralizing antibody blocked the HGF-induced decrease in cell proliferation. Cells treated with 100 ng/mL of HGF-neutralizing antibody plus HGF (40 ng/ mL) did not significantly differ from control cells but did proliferate significantly more than HGF-treated cells (P ⬍ .05). A higher dose of neutralizing antibody (300 ng/mL) in combination with HGF (40 ng/mL) also displayed a significantly higher cell proliferation than HGF-treated cells (P ⬍ .05) but also was significantly lower in cell proliferation than control (P ⬍ .05). To show that the effect of HGF on mineralization was through its receptor, c-MET, we treated hMSCs with PHA665752, a c-MET inhibitor, and then quantified calcium accumulation during mineralization. We observed a significant (P ⬍ .05), concentration-dependent (0.1–1 M) decrease in mineralization across days 10 and 17 compared with control (Figure 4, A and B). PHA665752 at a lower dose, 0.05 M, was able to significantly reduce mineralization at day 10 but did not maintain the attenuated mineralization through day 17. Blocking cMET activation with PHA665752 also caused a significant, dose-dependent decrease in alkaline phosphatase activity. Three days after PHA665752 treatment (0.1, 0.25, 0.5, and 1 M) hMSCs displayed reduced alkaline phosphatase activity (Figure 4 C). PHA665752 at a lower dose, 0.05 M, did not attenuate alkaline phosphatase activity 3 days after treatment.
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utes. Phosphorylation of p38 was not determined at this time due to the lack of a specific phospho-p38 antibody. HFG increases the level of p38␣ and p38 To determine whether HGF treatment on p38 signal transduction is isoform specific, we performed Western blot and RT-qPCR analyses on p38␣ and p38 isoforms. A single treatment of HGF (40 ng/mL) led to a transient increase in p38␣ and p38 in hMSCs. Western blot analysis showed an increase in both p38␣ and p38 on day 1. Both p38␣ and p38 maintained protein expression through days 2 and 3, with Figure 3. HGF neutralizing antibody reduces mineralization. hMSCs were treated with 0 (C), 5, p38 displaying the highest increase 10, 100, 200, or 300 ng/mL of HGF neutralizing antibody (R&D; MAB294) during media changes in protein expression compared every 3– 4 days and then stained using Alizarin Red-S on days 7 and 10 (panel A). Mineralization with control (Figure 6A). RT-qPCR was then quantified and absorbance measured at 550 nm (panel B). Cell proliferation assay of analysis of p38 gene expression after HGF neutralizing antibody (100 and 300 ng/mL) with or without HGF (40 ng/mL) at day 5 (panel C). Error bars represent SD. *, P ⬍ .05 vs control by Student’s t test; #, P ⬍ .05 vs HGF-treated a single HGF treatment revealed difusing Student’s t test. C, control. ferential expression of p38␣ and p38 (Figure 6B). HGF (40 ng/mL) did not significantly affect gene exWestern blot analysis showed that at doses of 0.1 and 1.0 M, PHA665752 did block HGF-induced activation of pression levels of p38␣ after 1 day of treatment. However, cMET (ie, phospho-cMET) (Figure 4D). Densitometric 2 days of HGF treatment did show a significant increase analysis showed that HGF alone produced an increase of in the gene expression of p38␣ (P ⬍ .05), followed by a 3.5 (350% increase) compared with control. Doses of 0.1 significant decrease (P ⬍ 0,05) in the gene expression of and 1.0 M of PHA665752 produced cMet activation of p38␣ at day 3. HGF significantly altered the level of 1.9 and 1.2 (PHA665752 ⫹HGF), roughly 50% and p38. On day 1 expression level of p38 gene was signif70% decreases in activation compared with HGF treated, icantly increased by HGF treatment (P ⬍ .05). The most robust response (P ⬍ .0001) to HGF treatment was seen respectively. at day 2, with a 10-fold increase in expression compared with basal levels of p38 expression. The gene expression HGF rapidly phosphorylates p38 in a dose- and time-dependent manner level of p38 returned to basal levels by day 3. In view of the reports cited above that p38 is involved in skeletogenesis, we performed a dose response and time p38␣ and p38 differentially regulate HGF-induced course of HGF treatment to determine whether HGF af- expression of key osteogenic markers siRNA was used to determine the role(s) of p38␣ and fects p38 signal transduction in hMSCs. HGF (40 ng/mL)  in HGF induction of osteogenic markers. Two days p38 treatment of hMSCs showed a significant increase in phosphorylation of total p38 and continued with 100 of HGF treatment in siControl-treated cells significantly ng/mL after 10 minutes of treatment (Figure 5A). HGF (P ⬍ .05) increased the transcription level of OPG and concentrations of 10 and 20 ng/mL did not significantly OSX compared with siControl, with a 5.5- and 1.5-fold increase p38 phosphorylation within 10 minutes of treat- (respectively) increase. A significant increase was not seen ment. A time course with 40 ng/mL HGF led to significant for OCN (P ⫽ .06), COL1a1, or BSP2 when comparing phosphorylation of p38␣ within 5 minutes and phosphor- siControl vs siControl ⫹HGF, although a significant inylation of total p38 within 10 minutes (Figure 5B). Phos- crease was seen in Figure 1. Compared with the results phorylation of both p38␣ and total p38 peaked at 10 –15 seen in Figure 1, Figure 7 displays the results after a total minutes and subsequently decreased at 30 and 60 min- of 6 days in culture. The change in HGF levels between
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receiving HGF. However, the increase in OPG was truncated compared with siControl and p38␣ siRNA treated with HGF; they displayed a roughly 5.5- and 3.7-fold, respectively, increase.
Discussion The data presented herein is the first account describing the interaction of HGF, p38␣, and p38 on osteogenic maturation. The HGF/p38 signal transduction pathway provides a novel mode of regulation/support for bone maintenance and bone renewal. HGF plays a vital role in bone maintenance and bone renewal through the activation of key osteoFigure 4. cMET inhibitor, PHA665752, blocks mineralization. hMSCs were treated with 0 (C), genic markers and promoting osteo0.05, 0.1, 0.25, 0.5, or 1 M cMET inhibitor (PHA665752) during media changes every 3– 4 days blast proliferation (1, 21). In paraland then stained using Alizarin Red-S on days 10 and 17 (panel A). Mineralization was then quantified and absorbance measured at 550 nM (panel B). Alkaline phophosphatase activity was lel, our data show that HGF induces determined at day 3 (panel C). Western blot analysis showed a reduction of phosphorylated cMet the expression of OCN, OPG, and total cMet (␣-tubulin as a loading control), with densitometry quantification shown (panel COL1a1, BSP2, and OSX in a tranD). Error bars represent SD. *, P ⬍ .05 vs control using Student’s t test. C, control. sient manner. Although gene expression of these key osteogenic markers day 2 and day 6 may be contributing to the lack of effect begins to rise in hMSC after 1 day of HGF treatment, we of HGF on siControl. When HGF is added to the MSCs on day 2, the level of HGF is still elevated and/or the rate observed the most robust effect at day 2, with the levels of turnover is lower than on day 6. It is possible that the then returning to baseline by day 3. Similar to our data, 40 ng/mL plus the elevated level of endogenous HGF is HGF (30 ng/mL) has also been reported to induce osteowhat is needed to drive the gene transcription of OCN pontin gene expression within 24 hours (21). Using a and other osteogenic markers. Also it is possible that on rabbit model of osteonecrosis, Wen et al (22) reported an day 6 the rate of turnover is too high for the 40 ng/mL of improvement in tissue repair when transplanted MSCs were transfected with HGF. In their model, HGF levels HGF to have an effect on siControl-treated cells. The results of HGF treatment in cells treated with ei- immediately increased following transplantation and did ther p38␣ siRNA or p38 siRNA show significant not decline until 2 weeks after transplantation. Recent changes in gene expression. RT-qPCR data reveal a sig- work by Tomson et al (23) showed an increase in OSX nificant (P ⬍ .05) increase in the expression of OCN, and OCN gene expression after 7 and 12 days of treatOPG, and COL1a1 when HGF-treated cells were exposed ment with HGF. In those studies experimental media to p38␣ siRNA. However, although the data show a were changed every 48 hours, suggesting a cumulative trend, HGF did not significantly increase the expression effect of HGF treatment after continued treatment (23). of OSX or BSP2 in p38␣ siRNA-exposed cells. Most im- Along these lines, we have previously reported that HGF portantly, HGF did not induce the expression of OCN, inhibits cell proliferation and, combined with 1␣,25-diOSX, or COL1a1 in cells in which the level of p38 was hydroxy vitamin D, promotes osteogenic differentiation decreased. Cells treated with p38 siRNA failed to re- (3, 18, 31). Thus it appears that both acute and chronic spond to HGF induction of gene expression, displaying a treatment with HGF promotes osteogenesis. Using chemical inhibition of the HGF pathway we saw similar level of transcriptional activity as cells treated with p38 siRNA without HGF. OPG and BSP2 did show a marked reduction in mineralization and alkaline phosa significant increase in gene expression in p38 siRNA phatase activity. hMSCs produce HGF, and endogenous HGF-treated cells compared with p38 siRNA cells not HGF can have autocrine and paracrine activity on the
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The role of p38 in bone development and bone maintenance is becoming widely recognized. p38␣-knockout mice have reduced bone mass, and mice lacking p38␣ in osteoblasts display an age-dependent decrease in bone mass and bone mineral density (12, 24). Similarly, p38knockout mice also exhibit a diminished bone mass (12). Greenblatt et al (12) hypothesized that p38␣ and p38 have different functions in bone development and maintenance: p38␣ regulating early osteoblast differentiation and p38 regulating late osteoblast maturation. Our data show that p38 appears to have a major role in the HGF induction of genes associated with mature osteoblasts (eg, OCN). For example, HGF induction of OCN and Col1a1 are dependent on p38. OCN, secreted solely by osteoblast, is necessary for alkaline phosphatase activity and mineralization. Additionally, HGF was not able to induce Figure 5. HGF activation of p38 is dose and time dependent. Dose OXS gene expression in p38␣- or p38 siRNA-treated response (panel A) of HGF treatment (10 minutes) of hMSCs. Time course cells. Therefore, OSX, a key mediator of early osteoblast (panel B) of HGF treatment (40 ng/mL) of hMSCs. Western blot analyses were performed on phosphorylated-p38␣ (p-p38␣), total p38␣ (p38␣), differentiation, appears to require both p38␣ and p38 phosphorylated-p38 (p-p38), and total p38 (p38), with ␣-tubulin as a for full HGF induction. The HGF-induced expression of loading control (n ⫽ 3 independent experiments in triplicate, with a OPG was truncated, but not removed, with either p38␣representative blot and densitometric quantification shown). or p38 siRNA. In contrast, BSP2 showed significant incells (3). Blocking the endogenous HGF activity, through crease in gene expression in p38 siRNA-treated cells but the inhibition of c-MET activity or with an antibody neu- not in p38␣ siRNA-treated cells. Hence, our data support tralizing endogenous HGF, was sufficient to attenuate the role of HGF and p38 in early and late stages of osteoosteogenic differentiation of hMSCs. These results sug- blast differentiation. gest that basal HGF activity is promoting bone mainteAlthough p38␣ is the most abundant p38 isoform (as nance/osteoblast differentiation. Although we did not see seen in our hMSCs, data not shown), the role of p38 is an increase in mineralization or alkaline phosphatase important for bone mass. As indicated above, p38(data not shown) using 40 ng/mL of HGF, it is likely due knockout mice display a reduction in bone mass (12). to a ceiling effect with the endogenous HGF (Figure 2B) Interestingly, p38␣ is the major isoform found in osteactivity combined with the differentiation media rapidly oclasts and is essential for osteoclast differentiation and pushing the cells toward maturation/mineralization and bone resorption (25). Our data suggest that p38 is necincreasing alkaline phosphatase activity, thus making it essary for the induction of gene expression of osteogenic difficult to see an increase in our system. markers, but it is unclear whether this is due to the loss of p38-mediated gene expression or whether p38␣ is acting as a direct inhibitor of genes involved in osteogenic differentiation. Moreover, the rapid activation of p38 by HGF also contributes to osteogenic differentiation. Here we show that HGF rapidly activates p38 via phosphorylation within 10 minutes of treatment. This effect is dose dependent, with at Figure 6. HGF increases the level of p38 protein (panel A) and mRNA expression (panel B). least 40 ng/mL of HGF needed to see Western blot analysis (panel A) of p38␣ and p38 after a single treatment with HGF (40 ng/mL) a significant increase in p38 phoson days 0, 1, 2, and 3. (n ⫽ 3 independent experiments in triplicate with a representative blot phorylation. HGF treatment also and densitometric quantification shown). B, RT-qPCR of p38 isoforms after HGF treatment (40 ng/mL) on days 0, 1, 2, and 3. Relative values were calculated by ⌬⌬CT method, with each gene leads to increases in p38␣ and p38 normalized to housekeeping gene RPL13␣ and its control (day 0). Error bars represent SD of protein levels and in their gene extriplicate experiments; *, P ⬍ .05 vs control; and **, P ⬍ .0001 vs control using Student’s t test. pression. It is likely that HGF is inControl has no error bar because the values obtained for each experiment were set at 1 for fluencing the expression of p38 subsequent fold change calculation.
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Figure 7. p38 and p38␣ mediate HGF-induced osteogenic gene expression. RT-qPCR was performed on RNA from hMSCs exposed to 4 days of siRNA control, siRNA p38␣, or siRNA p38. On day 4 the cells were then treated or not with HGF (40 ng/mL) for an additional 2 days (6 days total). Relative values were calculated by ⌬⌬CT method, with each gene normalized to housekeeping gene RPL13␣ and its control (day 0) (left panels and 2 lower right panels). Western blot analysis of the knockdown of p38␣ and p38 4 days after siRNA treatment (upper right panel). Error bars represent SD of 3 independent experiments; *, P ⬍ .05 vs control using Student’s t test. siControl has no error bar because the values obtained for each experiment were set at 1 for subsequent fold change calculation.
through the rapid phosphorylation of p38, and thereby maintaining the protein levels, ie, activated p38 is less likely to be degraded. Additionally, the increase in gene expression can lead to increased protein levels by days 2 and 3. HGF’s dual induction and maintenance of p38 activity appear to have numerous effects on hMSCs, including the induction of key osteogenic proteins. The p38 MAPK pathway is likely promoting osteoblast differentiation and maturation by acting as a mediator of both the activation of key osteogenic proteins and transcriptional activity of key osteogenic genes. Phosphorylation of p38 leads to downstream effects including the activation and expression of RUNX2 and OSX (26 – 29). Moreover, p38 plays a key role in OSX-induced expression of COL1a1 (30). Finally, p38 has been reported to be essential in mineralization and alkaline phosphatase activity during osteogenesis in MC3T3-E1 cells (31, 32), in which p38 promotes the transcription of osteogenic genes through posttranscriptional modification.
The role of p38 in HGF-mediated osteogenic differentiation and bone repair has promising therapeutic potential in that promotion of p38 over p38␣ might lead to a stronger HGF effect in osteogenic maturation and bone repair. The combination of p38 activity and HGF treatment in hMSCs may have important clinical applications for the treatment of bone diseases and the promotion of bone health.
Acknowledgments We thank David Vazquez and B. Nubia Rodriguez for excellent technical assistance. Address all correspondence to: Guy A. Howard, Veterans Affairs Medical Center GRECC (11GRC), 1201 NW 16th Street, Miami, FL 33125. E-mail: [email protected]. For reprints contact: [email protected]. This work was supported by Merit Review awards from the Department of Veterans Affairs (to B.A.R., G.A.H.) and the
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National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award 7F32AR062990 (to K.M.C.). Dr Howard is the recipient of a Senior Research Career Scientist award from the Department of Veterans Affairs. Disclosure Summary: The authors have nothing to disclose.
Mol Endocrinol, May 2014, 28(5):722–730
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