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We previously reported that transcription factor XBP1S binds to RUNX2 and enhances chondrocyte hypertrophy through acting as a cofactor of RUNX2. Herein, we report that XBP1S is a key downstream molecule of BMP2 and is required for BMP2-mediated chondrocyte differentiation. XBP1S is up-regulated during chondrocyte differentiation and demonstrates the temporal and spatial expression pattern during skeletal development. XBP1S stimulates chondrocyte differentiation from mesenchymal stem cells in vitro and endochondral ossification ex vivo. In addition, XBP1S activates granulin-epithelin precursor (GEP), a growth factor known to stimulate chondrogenesis, and endogenous GEP is required, at least in part, for XBP1S-stimulated chondrocyte hypertrophy, mineralization and endochondral bone formation. Furthermore, XBP1S enhances GEP-stimulated chondrogenesis and endochondral bone formation. Collectively, these findings demonstrate that XBP1S, a BMP2-inducible transcription factor, positively regulates endochondral bone formation by activating GEP chondrogenic growth factor.

BackgroundWe reported earlier that X-box binding protein1 spliced (XBP1S), a key regulator of the unfolded protein response (UPR), as a bone morphogenetic protein 2 (BMP2)-inducible transcription factor, positively regulates endochondral bone formation by activating granulin-epithelin precursor (GEP) chondrogenic growth factor. Under the stress of misfolded or unfolded proteins in the endoplasmic reticulum (ER), the cells can be protected by the mammalian UPR. However, the influence of activating transcription factor 6 (ATF6), another transcriptional arm of UPR, in BMP2-induced chondrocyte differentiation has not yet been elucidated. In the current study, we investigate and explore the role of ATF6 in endochondral bone formation, focus on associated molecules of hypertrophic chondrocyte differentiation, as well as the molecular events underlying this process.MethodsHigh-cell-density micromass cultures were used to induce ATDC5 and C3H10T1/2 cell differentiation into chondrocytes. Quantitative real-time PCR, immunoblotting analysis, and immunohistochemistry were performed to examine (1) the expression of ATF6, ATF6α, collagen II, collagen X, and matrix metalloproteinase-13 (MMP13) and (2) whether ATF6 stimulates chondrogenesis and whether ATF6 enhances runt-related transcription factor 2 (Runx2)-mediated chondrocyte hypertrophy. Culture of fetal mouse bone explants was to detect whether ATF6 stimulates chondrocyte hypertrophy, mineralization, and endochondral bone growth. Coimmunoprecipitation was employed to determine whether ATF6 associates with Runx2 in chondrocyte differentiation.ResultsATF6 is differentially expressed in the course of BMP2-triggered chondrocyte differentiation. Overexpression of ATF6 accelerates chondrocyte differentiation, and the ex vivo studies reveal that ATF6 is a potent stimulator of chondrocyte hypertrophy, mineralization, and endochondral bone growth. Knockdown of ATF6 via a siRNA approach inhibits chondrogenesis. Furthermore, ATF6 associates with Runx2 and enhances Runx2-induced chondrocyte hypertrophy. And, the stimulation effect of ATF6 is reduced during inhibition of Runx2 via a siRNA approach, suggesting that the promoting effect is required for Runx2.ConclusionsOur observations demonstrate that ATF6 positively regulates chondrocyte hypertrophy and endochondral bone formation through activating Runx2-mediated hypertrophic chondrocyte differentiation.

Bone morphogenetic protein 2 (BMP2) is known to activate unfolded protein response (UPR) signaling molecules, such as BiP (IgH chain-binding protein), PERK (PKR-like ER-resistant kinase), and IRE1α. Inositol-requiring enzyme-1a (IRE1a), as one of three unfolded protein sensors in UPR signaling pathways, can be activated during ER stress. Granulin-epithelin precursor (GEP) is an autocrine growth factor that has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation. However, the influence on IRE1a in BMP2-induced osteoblast differentiation has not yet been elucidated. Herein we demonstrate that overexpression of IRE1a inhibits osteoblast differentiation, as revealed by reduced activity of alkaline phosphatase (ALP) and osteocalcin; however, knockdown of IRE1a via the RNAi approach stimulates osteoblastogenesis. Mechanistic studies revealed that the expression of IRE1a during osteoblast was a consequence of JunB transcription factor binding to several AP1 sequence (TGAG/CTCA) in the 5′-flanking regulatory region of the IRE1a gene, followed by transcription. In addition, GEP induces IRE1a expressions and this induction of IRE1a by GEP depends on JunB. Furthermore, IRE1a inhibition of GEP-induced osteoblastogenesis relies on JunB. Besides, GEP is required for IRE1a inhibition of BMP2-induced bone formation. Collectively, these findings demonstrate that IRE1a negatively regulates BMP2-induced osteoblast differentiation and this IRE1a inhibition effect depends on GEP growth factor. Thus, IRE1a, BMP2, GEP growth factor, and JunB transcription factor form a regulatory loop and act in concert in the course of osteoblastogenesis.

Eukaryotic cells possess several mechanisms to adapt to endoplasmic reticulum (ER) stress and thereby survive. ER stress activates a set of signaling pathways collectively termed as the unfolded protein response (UPR). We previously reported that Bone morphogenetic protein 2 (BMP2) mediates mild ER stress and activates UPR signal molecules in chondrogenesis. The mammalian UPR protects the cell against the stress of misfolded proteins in the endoplasmic reticulum. Failure to adapt to ER stress causes the UPR to trigger apoptosis. Glucose regulated protein 78 (GRP78), as an important molecular chaperone in UPR signaling pathways, is responsible for binding to misfolded or unfolded protein during ER stress. However the influence on GRP78 in BMP2-induced chondrocyte differentiation has not yet been elucidated and the molecular mechanism underlyng these processes remain unexplored. Herein we demonstrate that overexpression of GRP78 enhanced cell proliferation in chondrocyte development with G1 phase advance, S phase increasing and G2-M phase transition. Furthermore, overexpression of GRP78 inhibited ER stress-mediated apoptosis and then reduced apoptosis in chondrogenesis induced by BMP2, as assayed by cleaved caspase3, caspase12, C/EBP homologous protein (CHOP/DDIT3/GADD153), p-JNK (phosphorylated c-Jun N-terminal kinase) expression during the course of chondrocyte differentiation by Western blot. In addition, flow cytometry (FCM) assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay and immune-histochemistry analysis also proved this result in vitro and in vivo. It was demonstrated that GRP78 knockdown via siRNA activated the ER stress-specific caspase cascade in developing chondrocyte tissue. Collectively, these findings reveal a novel critical role of GRP78 in regulating ER stress-mediated apoptosis in cartilage development and the molecular mechanisms involved.