ATF6 is activated in response to ER stress.

Transmission of ER stress response by ATF6 promotes endochondral bone growth.

“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.
High-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.
ATF6 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{ATF6 may increase height, sometimes accelerators of growth increase height sometimes not}. 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.
Our observations demonstrate that ATF6 positively regulates chondrocyte hypertrophy and endochondral bone formation through activating Runx2-mediated hypertrophic chondrocyte differentiation.”

“BMP2 can activate unfolded protein response (UPR)-signaling molecules, such as BiP (binding immunoglobulin protein), CHOP (C/EBP homologous protein), ATF4 (activating transcription factor 4), and IRE1α (inositol-requiring enzyme-1α). ”

“The UPR is divided into three arms, including the PKR-like ER-resistant kinase (PERK), activating transcription factor 6 (ATF6), and IRE1α; the three together act to restrict new protein synthesis and increase the production of chaperones.”

“. BMP2 induces mild ER stress, and then ATF6, as a 90-kDa protein (p90ATF6) in previous non-ER stress environment, is directly converted to a 50-kDa protein (p50ATF6, ATF6a) in ER-stressed cells.  ATF6 undergoes proteolysis and splicing after BMP2 stimulation. ATF6a protein was not detected until day 5 in BMP2-induced chondrocyte differentiation of ATDC5 cells. The expression of collagen X was also immune positive at day 7, indicating that ATF6a expression is prehypertrophic and hypertrophic chondrocyte-specific. The ER stress-induced ATF6 proteolysis occurs in BMP2 stimulation day 5. More significantly, ATF6a expression was 2 days earlier than that of collagen X.”

“ATF6 significantly stimulated chondrocyte hypertrophy, mineralization, and bone length.”

“ATF6 associates with Runx2 in chondrogenesis and ATF6 enhances Runx2-mediated chondrocyte hypertrophy”