Abstract
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.
Highlights
We 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
ATF6a protein was not detected until day 5 in BMP2-induced chondrocyte differentiation of ATDC5 cells
Knockdown of activating transcription factor 6 (ATF6) obviously abolished the collagen II (Fig. 6b, f ), collagen X (Fig. 6c, g), and matrix metalloproteinase-13 (MMP13) (Fig. 6d, h) induction during chondrocyte differentiation. These findings clearly indicated that knockdown of ATF6 via small interfering RNA (siRNA) clearly inhibited BMP2induced chondrocyte differentiation and hypertrophy, and endogenous ATF6 is required for hypertrophic chondrocyte differentiation
Summary
We reported earlier that X-box binding protein 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. The influence of activating transcription factor 6 (ATF6), another transcriptional arm of UPR, in BMP2-induced chondrocyte differentiation has not yet been elucidated. 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. X-box binding protein spliced (XBP1S) positively regulates endochondral bone formation by activating granulin-epithelin precursor (GEP) chondrogenic growth factor [13]. Whether ATF6 can influence the chondrocyte differentiation and endochondral bone growth has not yet been elucidated; the molecular mechanism underlying these processes remains unexplored. Our results support a novel role of ATF6 and endochondral bone growth through Runx transcription factor
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