HSPB7 regulates osteogenic differentiation of human adipose derived stem cells via ERK signaling pathway

Heat shock protein B7 (HSPB7), which belongs to small heat shock protein family, has been reported to be involved in diverse biological processes and diseases. However, whether HSPB7 regulates osteogenic differentiation of human adipose derived stem cells (hASCs) remains unexplored. The expression level of HSPB7 during the osteogenesis of hASCs was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis. Lentivirus transfection was used to knock down or overexpress HSPB7, which enabled us to investigate the effect of HSPB7 on osteogenic differentiation of hASCs. U0126 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) siRNA were used to identify the mechanism of the HSPB7/ERK1/2 axis in regulating osteogenic differentiation of hASCs. Moreover, ectopic bone formation in nude mice and osteoporosis mice model was used to investigate the effect of HSPB7 on osteogenesis in vivo. In this study, we found the expression of HSPB7 was significantly downregulated during the osteogenic differentiation of hASCs. HSPB7 knockdown remarkably promoted osteogenic differentiation of hASCs, while HSPB7 overexpression suppressed osteogenic differentiation of hASCs both in vitro and in vivo. Moreover, we discovered that the enhancing effect of HSPB7 knockdown on osteogenic differentiation was related to the activation of extracellular signal-regulated protein kinase (ERK) signaling pathway. Inhibition of ERK signaling pathway with U0126 or silencing ERK1/2 effectively blocked the stimulation of osteogenic differentiation induced by HSPB7 knockdown. Additionally, we found that HSPB7 expression was markedly increased in mouse bone marrow mesenchymal stem cells (mBMSCs) from the osteoporotic mice which suggested that HSPB7 might be utilized as a potential target in the development of effective therapeutic strategies to treat osteoporosis and other bone diseases. Taken together, these findings uncover a previously unrecognized function of HSPB7 in regulating osteogenic differentiation of hASCs, partly via the ERK signaling pathway.


Introduction
Human adipose derived stem cells (hASCs) are multipotential mesenchymal stem cells (MSCs) that have the ability to differentiate into different lineages such as chondrocytes, adipocytes, and osteocytes [1]. As a type of MSCs, hASCs offer new therapeutic solutions for bone defects and metabolic diseases due to their abundant sources, ease of acquisition, low immunogenicity, and osteogenic differentiation capacity [2,3]. However, the underlying mechanisms by which hASCs undergo osteogenic differentiation are not yet fully elucidated, thereby hindering their potential clinical applications.
Heat shock protein B7 (HSPB7), also known as cardiovascular heat shock protein (cvHsp), belongs to the small heat shock protein family with essential roles in maintaining cellular homeostasis in a variety of both physiological and pathophysiological processes [13][14][15]. HSPB7 is highly expressed in the cardiac tissue and plays a critical role in cardiac metabolism [16]. The dysfunction of HSPB7 is strongly associated with heart failure, dilated cardiomyopathy, and idiopathic cardiomyopathy [17]. HSPB7 has also been demonstrated to function as a tumor suppressor in multiple malignancies such as renal cell carcinoma [18,19]. However, the effects of HSPB7 on osteogenic differentiation still remain unclear.
In this study, we evaluated the function of HSPB7 in the osteogenic differentiation of hASCs both in vitro and in vivo. We found HSPB7 knockdown promoted osteogenic differentiation of hASCs via activation of the ERK signaling pathway. These findings could contribute to better understanding of the molecular mechanisms underlying osteogenic differentiation and provide new therapeutic target for treating bone diseases.

Alkaline phosphatase (ALP) staining
Cells were cultured in PM or OM for 7 days. After that, cells were washed with PBS, fixed in 4% paraformaldehyde for 15 min, incubated with the ALP substrate solution (CoWin Biotech, Beijing, China), and recorded by a scanner (Image Scanner III, GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA).

Quantification of ALP activity
Cells were washed with PBS, treated with 1% Triton X-100 (Sigma), and assayed with the ALP Activity Kit (Biovision, Milpitas, CA). ALP activity was quantified by reading the absorbance at 520 nm and normalized to the total protein content. Total protein concentration was determined using a BCA protein assay kit (Pierce Thermo Scientific, Waltham, MA, USA).

Alizarin Red S (ARS) staining and quantification
At 14 days after osteogenic induction, cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, and rinsed with Milli-Q water. Next, the calcium deposition was stained with 1% Alizarin Red S solution (pH 4.2, Sigma) for 20 min. For quantification, the stain was dissolved in cetylpyridinium chloride (Sigma) and measured with a spectrophotometer at 562 nm.

Oil Red O staining
To induce adipocyte differentiation, hASCs were cultured in adipocyte differentiation medium (AM) consisting of standard PM supplemented with 50 nM insulin (Sigma), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), 100 nM dexamethasone (Sigma), and 200 mM indomethacin (Sigma). At day 14 after adipogenic induction, cells were washed twice with PBS, fixed with 10% formalin for 30 min, and stained with Oil Red O working solution (0.3%, Sigma). Then, the stained cells were washed with distilled water and recorded by a microscope.

Immunofluorescence staining
Cells grown on the glass coverslips were washed with ice-cold PBS, fixed with 4% formaldehyde, permeabilized with 0.2% Triton X-100, and then blocked with 1% bovine serum albumin. Next, cells were incubated with primary antibody against OCN (Abcam) at 4°C overnight and then incubated with a fluorescence conjugated secondary antibody for 60 min at room temperature. After washing with PBS, cells were stained with DAPI for detection of nuclei. Images were visualized using a confocal microscope (Carl Zeiss, Oberkochen, Germany).

RNA isolation and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis
Total RNA was extracted using the TRIzol Reagent (Invitrogen) following the manufacturer's instructions.
The reverse transcription was performed with the Pri-meScript RT Reagent Kit (Takara, Tokyo, Japan). qRT-PCR was implemented with SYBR Green Master Mix (Roche Applied Science, Mannheim, Germany) on a 7500 Real-Time PCR Detection system (Applied Biosystems, Foster City, CA, USA) with gene-specific primers. The relative expression levels were measured by comparative cycle threshold (CT) method.
The primer sequences used are as follows: HSPB7,

Ectopic bone formation in vivo
All animal protocols were performed in accordance with the laboratory animal care and use guidelines and approved by the Peking University Animal Care and Use Committee. Thirty BALB/c homozygous nude (nu/nu) male mice (8 weeks, 18-20 g) purchased from Vital Co. (Beijing, China) were randomly divided into five groups (n = 6 per group). Cells were induced in OM for 7 days, resuspended, incubated with Bio-Oss Collagen (Geistlich, GEWO GmbH, Baden-Baden, Germany) for 1 h at 37°C, and then implanted into the dorsal subcutaneous sites of nude mice. After 8 weeks, the implants were harvested and fixed with 4% formalin for 24 h, decalcified in 10% EDTA for 14 days, and followed by embedding in paraffin. Sections (5 μm thick) were cut and stained with hematoxylin and eosin (HE) and Masson's trichrome.

Establishment of ovariectomized mouse model
Eight-week-old female BALB/c mice weighting 20 ± 2 g were obtained from Vital Co. After 1 week of acclimatization, the mice were randomly allocated to two groups: sham operation group (SHAM) and ovariectomized group (OVX). The ovariectomy was performed as described previously [20]. Twelve weeks following the ovariectomy surgery, femurs were collected and evaluated using a micro-computed tomography (micro-CT) system. The scanner was set at a current of 220 μA, a voltage of 60 kV, and a resolution of 9.088 μm per pixel. Inveon Research Workplace (Inveon, Siemens, Munich, Germany) was used to calculate morphometric parameters, including bone volume/total volume (BV/TV), trabecular separation (Tb.Sp), and trabecular number (Tb.N) in the trabecular region (1 to 2 mm distal to the proximal epiphysis). Then, the femoral specimens were decalcified in 10% EDTA solution, embedded in paraffin, and stained with hematoxylin and eosin (HE). mBMSCs were collected from the tibias of SHAM and OVX mice as described previously [21].

Statistical analysis
All data were expressed as mean ± standard deviation (SD). The data were calculated with SPSS statistics version 16.0 (SPSS Inc., Chicago, IL, USA). Independent sample t test was used for comparison of two groups, and one-way analysis of variance (ANOVA) was applied for the comparison among multiple groups. A value of P < 0.05 was considered statistically significant.

HSPB7 was downregulated during osteogenic differentiation of hASCs
In order to determine the effect of HSPB7 in the process of osteogenesis, we first detected the expression level of HSPB7 during osteogenic differentiation of hASCs. qRT-PCR analysis showed the mRNA expression of HSPB7 was remarkably decreased during osteogenesis, while the expression levels of osteogenic markers RUNX2, ALP, and OCN were significantly upregulated (Fig. 1a-d). Consistently, Western blot revealed a similar trend in protein expression (Fig. 1e, f).
Moreover, we compared the expression of HSPB7 among hASCs, hBMSCs, and human osteoblast-like U2OS cells. Results showed that HSPB7 was expressed at a much lower level in U2OS cells compared with hASCs and hBMSCs (SFig. 1A). Meanwhile, we compared the expression level of Hspb7 between mBMSCs and mouse MC3T3-E1 cells and found that the level of Hspb7 was very low in mouse MC3T3-E1 cells compared with mBMSCs (SFig. 1B). These results indicated that HSPB7 might be associated with the stage of osteogenic differentiation. Additionally, we found long-term expansion of hASCs in vitro affected the expression of HSPB7. As shown in SFig. 1C, hASCs at late passages have a higher expression of HSPB7 compared with hASCs at early passages.

HSPB7 overexpression inhibited osteogenic differentiation of hASCs
To clarify whether HSPB7 regulates osteogenic differentiation of hASCs, we overexpressed HSPB7 in hASCs by lentivirus transfection. Fluorescent staining revealed the transfection efficiency was more than 90% (SFig. 2A). Western blot and qRT-PCR showed the expression level of HSPB7 increased by more than 15 folds in HSPB7 overexpression hASCs in comparison to the negative control hASCs (NC) (SFig. 2B-D). After culturing the infected hASCs in OM for 7 days, ALP activity was inhibited in HSPB7 overexpression cells compared to NC cells, as revealed by ALP staining and quantitative ALP activity assay (Fig. 2a, b). Similarly, extracellular matrix mineralization was also decreased in the HSPB7 overexpression group, as determined by ARS staining and quantification on day 14 (Fig. 2c, d). Furthermore, qRT-PCR was performed to detect the mRNA expression levels of osteogenic markers, which showed that HSPB7 overexpression downregulated the expression of RUNX2, ALP, and OCN after 14 days of osteogenic induction (Fig. 2e-g). Additionally, Western blot and immunofluorescence showed the protein expression of OCN was reduced in the HSPB7 overexpression group (Fig. 2h-j).

HSPB7 knockdown enhanced osteogenic differentiation of hASCs
The effect of HSPB7 on osteogenesis was further evaluated by gene knockdown experiments using shRNA. To avoid off-target effect, two different shRNA sequences against HSPB7 were employed. Following puromycin selection, the knockdown efficiency was verified by fluorescence staining (SFig. 2A), qRT-qPCR, and Western blot (SFig. 2E-G). As shown in Fig. 3a, b, ALP activity was significantly increased in the HSPB7 knockdown group compared to the shNC group. Consistently, a greater number of mineralized nodules were observed in the HSPB7 knockdown group than in the shNC group (Fig. 3c, d). Also, the mRNA expression levels of RUNX2, ALP, and OCN were increased after HSPB7 knockdown ( Fig. 3e-g). Additionally, HSPB7 knockdown upregulated the protein expression of OCN (Fig. 3h-j).
To further validate the function of HSPB7 in osteogenic differentiation, the recombinant human HSPB7 protein (rhHSPB7) was utilized for rescue experiments. Results showed the addition of rhHSPB7 successfully attenuated the osteogenic differentiation capacity of HSPB7 knockdown cells as well as the shNC cells (SFig. 3A-G). In addition, we also evaluated the effect of HSPB7 on Fig. 1 HSPB7 was downregulated during the osteogenic differentiation of hASCs. a Relative mRNA expression of HSPB7 was measured by qRT-PCR at days 0, 1, 4, 7, 10, and 14 during the osteogenic differentiation of hASCs. b-d Relative mRNA expression of RUNX2, ALP, and OCN during the osteogenic differentiation of hASCs. e After osteogenic induction, the protein expression of HSPB7 was significantly reduced, accompanied by the increased expression of RUNX2. f The quantitative results of e by Image J software. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control. Data are presented as the mean ± SD (*P < 0.05, **P < 0.01, compared with day 0) osteogenic differentiation of hBMSCs. Similarly, HSPB7 knockdown promoted osteogenesis of hBMSCs (SFig. 4A-F). Collectively, these findings suggested that HSPB7 played a negative role in osteogenic differentiation.

HSPB7 knockdown activated ERK1/2 signaling pathway
To further explore the mechanism by which HSPB7 regulates osteogenic differentiation, we examined several osteogenesis-related signaling pathways and found the expression of FGFR1 and p-ERK1/2 was significantly upregulated after HSPB7 knockdown (Fig. 4a-c). By contrast, the protein level of p-ERK1/2 was markedly decreased in HSPB7 overexpressing hASCs (SFig. 5A and B). Due to the abovementioned results, we hypothesized that ERK signaling might be involved in HSPB7mediated osteogenic differentiation. HSPB7 knockdown promoted osteogenesis through ERK1/ 2 signaling pathway To verify whether HSPB7 regulated osteogenesis via ERK1/2 signaling pathway, we examined the inhibitory effect of ERK1/2 signaling on osteogenic differentiation in the HSPB7 knockdown group. Western blot showed the level of p-ERK1/2 was significantly inhibited with the addition of U0126 (Fig. 5a, b). Following treatment with U0126, the increased ALP activity induced by HSPB7 knockdown was effectively abrogated (Fig. 5c, d). Meanwhile, the extracellular matrix mineralization was also reduced in HSPB7 knockdown hASCs in the presence of U0126 (Fig. 5e, f). Moreover, the upregulated mRNA expression levels of osteogenic markers RUNX2, ALP, and OCN caused by HSPB7 silencing were diminished with the addition of U0126 (Fig. 5g-i). Next, ERK1/2 siRNA was used to confirm whether ERK1/2 knockdown could block the increased osteogenic ability induced by HSPB7 deficiency. The knockdown efficiency of ERK1/2 was demonstrated by Western blot (SFig. 6A and B). As expected, the promotive effect of HSPB7 knockdown on osteogenesis was markedly reversed in the HSPB7 and ERK1/2 double knockdown hASCs, which was revealed by ALP staining and quantification (SFig. 6C and D), ARS staining and quantification (SFig. 6E and F), and qRT-PCR (SFig. 6G-I). Taken together, these results suggested that HSPB7 regulated osteogenic differentiation through ERK signaling pathway.
Although we found HSPB7 regulated osteogenesis with an involvement of ERK signaling pathway, we cannot exclude the possibility that other signaling pathways are also involved in HSPB7-mediated osteogenic differentiation. In this study, we also found a negative correlation between the mRNA expression of HSPB7 and BMPRI/II (SFig. 7). These data suggested that BMPRI/II might be implicated in HSPB7-regulated osteogenesis. Future investigation is needed to elucidate the mechanism.

HSPB7 inhibited bone formation of hASCs in vivo
To validate our findings in vitro, we examined whether HSPB7 could affect the bone formation ability of hASCs in vivo. As shown in Fig. 6a, HE staining and Masson's trichrome staining revealed the amount of newly generated bone was higher in HSPB7 knockdown hASCs compared with the control cells. On the contrary, mice implanted with HSPB7 overexpression hASCs showed much less bone-like tissue (Fig. 6b). Furthermore, quantitative measurements using SPOT 4.0 software confirmed that the volume of newly formed bone was increased in HSPB7 knockdown hASCs but reduced in HSPB7 overexpression hASCs (Fig. 6c). These results indicated that HSPB7 might suppress osteogenic differentiation of hASCs in vivo.

HSPB7 promoted adipogenesis of hASCs
It has been reported that osteogenesis and adipogenesis of MSCs are competing and reciprocal [22]. Thus, we also investigated the role of HSPB7 in adipogenesis. We observed that HSPB7 was upregulated during adipogenic differentiation of hASCs (SFig. 8A). Oil Red O staining and qRT-PCR showed that HSPB7 overexpression significantly promoted adipogenic differentiation of hASCs (SFig. 8B-D).

HSPB7 was increased in OVX mice
The imbalance between osteogenesis and adipogenesis is associated with various bone-related diseases, such as osteoporosis. Based on above results, it was reasonable to hypothesize that HSPB7 might be abnormally expressed in osteoporotic mice. To verify this hypothesis, we constructed osteoporotic mouse models by ovariectomy. HE staining and micro-CT showed that the trabecular number (Tb.N) and bone volume/total volume (BV/TV) were significantly reduced, whereas the trabecular separation (Tb.SP) was increased in OVX mice compared to those in SHAM mice, which confirmed the establishment of osteoporotic models (SFig. 9A-D). Then, mBMSCs were collected from OVX and SHAM mice and identified by flow cytometry. The protein expression of HSPB7 was greatly increased in mBMSCs from osteoporotic mice, as revealed by Western blot (SFig. 9E and F). These data suggested that HSPB7 might be involved in the pathogenesis of osteoporosis. More experimental studies are needed to further explore the role of HSPB7 in osteoporosis. DMSO was used as a control. Data are presented as the mean ± SD (*P < 0.05, **P < 0.01, compared with shNC)

Discussion
During recent years, hASCs isolated from adipose tissue have emerged as an attractive cell source for bone tissue engineering which sheds a light on bone repair and regeneration [23][24][25]. However, employing hASCs to promote bone repair is far from being applied in clinical routine due to the limited knowledge about osteogenic differentiation. Thus, it is of great significance to investigate the molecular basis of osteogenesis. Herein, we demonstrated that HSPB7 was markedly reduced during osteogenesis of hASCs and HSPB7 knockdown enhanced osteogenic capacity of hASCs both in vitro and in vivo.
HSPB7, located on 1p36.13, is a member of heat shock protein (HSP) family that plays essential roles in protein folding and cellular protein homeostasis [26]. Heat shock Fig. 6 HSPB7 knockdown promoted bone formation of hASCs in vivo. a HSPB7 knockdown promoted bone formation capacity of hASCs in vivo, as determined by HE staining (HE) and Masson's trichrome staining (Masson). b HSPB7 overexpression inhibited bone formation capacity of hASCs. c Quantitative measurements of bone-like tissue showed the area of bone-like tissue was significantly increased in HSPB7 knockdown hASCs but decreased in HSPB7 overexpression hASCs. Black arrows indicate new bone-like tissue. Scale bar = 50 μm. Data are presented as the mean ± SD (**P < 0.01, compared with shNC or NC) proteins have been implicated in diverse range of cellular processes, such as stress response, cell proliferation, and apoptosis [27][28][29]. Recent studies have revealed that heat shock proteins also play crucial roles in osteogenic differentiation. Flanagan et al. reported that heat shock protein B8 (HSPB8) knockdown significantly reduced osteogenic differentiation potential of dental pulp stem cells [30]. Chen et al. showed extracellular heat shock protein 70 (HSP70) enhanced osteogenesis of human mesenchymal stem cells (hMSCs) [31]. A study by Zhang et al. demonstrated low-intensity pulsed ultrasound stimulated osteogenesis of hASCs possibly by increasing the expression of HSP70 and HSP90 [32]. HSPB7, which is highly expressed in the heart, has been intensively investigated in multiple cardiac pathologies [33]. In the present study, our results provided novel insight into how HSPB7 regulated osteogenic differentiation of hASCs. We first showed that HSPB7 knockdown enhanced hASC osteogenesis through ERK signaling.
Extracellular signal-regulated kinase (ERK) pathway, also known as the Ras-Raf-MEK-ERK pathway, is involved in a variety of physiological processes such as cell proliferation, division, and survival [34,35]. Numerous studies have revealed that ERK signaling pathway plays vital roles in the osteogenic differentiation [36][37][38][39]. With regard to the relationship between HSPB7 and ERK signaling pathway, Naderi found HSPB7 overexpression in breast cancer cells reduced the level of p-ERK [18]. In agreement with the above study, we found HSPB7 overexpression reduced the level of p-ERK, while HSPB7 knockdown significantly activated ERK signaling pathway. To clarify the involvement of ERK signaling pathway in HSPB7-mediated osteogenic differentiation of hASCs, we examined whether inhibiting ERK signaling pathway with U0126 or siERK1/2 could attenuate the effect of HSPB7 knockdown on osteogenesis of hASCs. Results showed inhibition of ERK signaling pathway effectively blocked the enhancing effect of HSPB7 knockdown on osteogenesis. Although our study demonstrated that ERK signaling pathway played a crucial role in osteogenesis induced by HSPB7 knockdown, it is likely that other signaling pathways are also involved in this process. Previous studies indicated that HSPB7 was associated with diverse important signaling pathways. For example, Naderi found HSPB7 overexpression could downregulate the expression of p-AKT which is closely associated with osteogenic differentiation [18]. In this study, we observed the expression of BMPRI/II was negatively regulated by HSPB7, which suggested that BMPRI/II might be involved in HSPB7-regulated osteogenesis. Future studies are still needed to elucidate the mechanism of HSPB7-mediated osteogenesis.
Osteoporosis is a major public health problem which has a high incidence and great risk of fracture. To date, molecular pathways involved in the pathogenesis of osteoporosis remain elusive. Uncovering the mechanisms underlying osteoporosis is important for developing novel therapies and effective prevention strategies [40]. It has been well established that the imbalance between adipogenesis and osteogenesis contributes to osteoporosis [41]. In this study, we found HSPB7 expression was significantly increased during adipogenic differentiation of hASCs. We hypothesized that HSPB7 might be aberrantly expressed in osteoporosis model. Therefore, we constructed OVX mice and examined HSPB7 expression in mBMSCs from OVX. As expected, we found the protein expression of HSPB7 was significantly increased in OVX mice, which suggested the potential involvement of HSPB7 in the pathogenesis of osteoporosis. The present study provides new insight into the Fig. 7 Schematic diagram of the regulation of osteogenesis by HSPB7. HSPB7 regulated the osteogenic differentiation of hASCs via ERK1/2 signaling molecular basis of osteoporosis and implied that HSPB7 might be used as a potential diagnostic marker and therapeutic target for osteoporosis.

Conclusions
In summary, this is the first study to identify the negative role of HSPB7 in osteogenic differentiation of hASCs with an involvement of ERK signaling pathway (Fig. 7). These findings could provide not only new ideas for exploring new approaches in stimulating osteogenesis of hASCs but also a potential therapeutic target for the skeletal diseases.