Rong Jiang

ATF6 upregulates XBP1S and inhibits ER stress-mediated apoptosis in osteoarthritis cartilage

As we previously reported, transcription factor XBP1S enhances BMP2-induced chondrocyte differentiation and acts as a positive mediator of chondrocyte hypertrophy. The purpose of this study was to determine (1) whether XBP1S influences ER stress-mediated apoptosis in osteoarthritis (OA); (2) whether ATF6 regulates IRE1/XBP1 signal pathway in OA cartilage; (3) what are the associated molecules affecting apoptosis in osteoarthritis and the molecular events underlying this process. Herein, we examined and found that ER stress-associated molecules were activated in OA patients, specifically XBP1S splice and expression were increased markedly by TNF-α and IL-1β treatments. Transcription factor ATF6 can specifically bind to the promoter of XBP1 gene and enhance the expression of XBP1S spliced by IRE1α in osteoarthritis cartilage. Furthermore, siXBP1S can enhance ER stress-mediated apoptosis and main matrix degradation in osteoarthritis. Whereas AdXBP1S can inhibit ER stress-mediated apoptosis and TNFα induced nitrite production in OA cartilage. In a word, our observations demonstrate the importance of XBP1S in osteoarthritis. ATF6 and IRE1α can regulate endogenous XBP1S gene expression synergistically in OA cartilage. More significantly, XBP1S was a negative regulator of apoptosis in osteoarthritis by affecting caspase 3, caspase 9, caspase 12, p-JNK1, and CHOP.

Progranulin is required for proper ER stress response and inhibits ER stress-mediated apoptosis through TNFR2.

Progranulin (PGRN) was reported to be a stress-response factor in response to hypoxia and acidosis. Here we present evidences demonstrating that PGRN is also an endoplasmic reticulum (ER) stress responsive factor: PGRN expression was induced and its activation of Erk1/2 and Akt signaling enhanced in response to ER stress; Normal ER stress response was lost in PGRN deficient cells and PGRN deficient cells became hypersusceptible to ER stress-induced apoptosis; additionally, recombinant PGRN could rescue the defects in ER-stress responses seen in PGRN deficient cells. Mechanistic studies indicated that PGRN/TNFR2 was critical for PGRN mediated regulation of ER stress response: similar to PGRN, the expression of TNFR2, but not TNFR1, was also induced in the course of ER stress; in addition, the association between PGRN and TNFR2 was markedly enhanced following ER stress; More importantly, PGRN protection of ER stress induced apoptosis was abolished when TNFR2 signaling was blocked. In addition, the 2nd and 3rd cysteine-rich domains (CRD) in the extracellular portion of TNFR2 (CRD2CRD3), known to directly bind to PGRN, disturbed the interaction of PGRN with TNFR2, and in turn abolished PGRN-mediated activation of Erk1/2 and Akt signaling and protection against apoptosis in response to ER-stress. Collectively, PGRN plays an important role in ER stress and regulates ER stress response through interacting with TNFR2. This study provides new insight into PGRN regulation of stress response and may also present PGRN as a potential molecular target for treating stress-associated disorders.

IRE1α dissociates with BiP and inhibits ER stress-mediated apoptosis in cartilage development

Bone morphogenetic protein 2 is known to activate unfolded protein response signaling molecules, including XBP1S, BiP and IRE1α. Endoplasmic reticulum stress is induced in chondrogenesis and activates IRE1α signal pathway, which is associated with ER stress-mediated apoptosis. However, the influence on IRE1α and BiP in BMP2-induced chondrocyte differentiation has not yet been elucidated; the molecular mechanism remains unexplored. In this study, we demonstrate that IRE1α interacts with BiP in unstressed cells and dissociates from BiP in the course of cartilage development. Induction of ER stress-responsive proteins (XBP1S, IRE1α, BiP) was also observed in differentiating cells. IRE1α inhibition ER stress-mediated apoptosis lies in the process of chondrocyte differentiation. Furthermore, knockdown of IRE1α expression by way of the RNAi approach accelerates ER stress-mediated apoptosis in chondrocyte differentiation induced by BMP2, as revealed by enhanced expressions of cleaved caspase3, CHOP and p-JNK1; and this IRE1α inhibition effect on ER stress-mediated apoptosis is required for BiP in chondrogenesis. Collectively, the ER stress sensors were activated during apoptosis in cartilage development, suggesting that selective activation of ER stress signaling was sufficient for induction of apoptosis. These findings reveal a novel critical role of IRE1α in ER stress-mediated apoptosis and the molecular mechanisms involved. These results suggest that activation of p-JNK1, caspase3 and CHOP was detected in developing chondrocytes and that specific ER stress signaling leads to naturally occurring apoptosis during cartilage development.

XBP1S Associates with RUNX2 and Regulates Chondrocyte Hypertrophy

BMP2 (bone morphogenetic protein 2) is known to activate unfolded protein response signaling molecules, including XBP1S and ATF6. However, the influence on XBP1S and ATF6 in BMP2-induced chondrocyte differentiation has not yet been elucidated. In this study, we demonstrate that BMP2 mediates mild endoplasmic reticulum stress-activated ATF6 and directly regulates XBP1S splicing in the course of chondrogenesis. XBP1S is differentially expressed during BMP2-stimulated chondrocyte differentiation and exhibits prominent expression in growth plate chondrocytes. This expression is probably due to the activation of the XBP1 gene by ATF6 and splicing by IRE1a. ATF6 directly binds to the 5′-flanking regulatory region of the XBP1 gene at its consensus binding elements. Overexpression of XBP1S accelerates chondrocyte hypertrophy, as revealed by enhanced expression of type II collagen, type X collagen, and RUNX2; however, knockdown of XBP1S via the RNAi approach abolishes hypertrophic chondrocyte differentiation. In addition, XBP1S associates with RUNX2 and enhances RUNX2-induced chondrocyte hypertrophy. Altered expression of XBP1S in chondrocyte hypertrophy was accompanied by altered levels of IHH (Indian hedgehog) and PTHrP (parathyroid hormone-related peptide). Collectively, XBP1S may be a novel regulator of hypertrophic chondrocyte differentiation by 1) acting as a cofactor of RUNX2 and 2) affecting IHH/PTHrP signaling.

Explore on the effect of ATF6 on cell growth and apoptosis in cartilage development

We previously report that BMP2 mediates mild ER stress-activated ATF6 and directly regulates XBP1S splicing in the course of chondrogenesis. The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). Failure to adapt to ER stress causes the UPR to trigger apoptosis. The transcription factor activating transcription factor 6 (ATF6), a key regulator of the UPR, is known to be important for ER stress-mediated apoptosis and cell growth, but the molecular mechanism underlying these processes remains unexplored. In this study, we demonstrate that ATF6 is differentially expressed during BMP2-stimulated chondrocyte differentiation and exhibits prominent expression in growth plate chondrocytes. ATF6 can enhance the level of IRE1a-spliced XBP1S protein in chondrogenesis. IRE1a and ATF6 can synergistically regulate endogenous XBP1S gene expression in chondrogenesis. Furthermore, overexpression ATF6 inhibited, while ATF6-knockdown enhanced, the cell proliferation in chondrocyte development with G1 phase arresting, S phase reducing and G2-M phase delaying. Besides, Ad-ATF6 can activate, whereas knockdown ATF6 by an siRNA-silencing approach inhibited, ER stress-mediated apoptosis in chondrogenesis induced by BMP2, as assayed by cleaved caspase3, CHOP, p-JNK expression in the course of chondrocyte differentiation. On the other hand, FCM, TUNEL assay and immunohistochemistry analysis also proved this result in vitro and in vivo. It was demonstrated that Ad-ATF6 activation of the ER stress-specific caspase cascade in developing chondrocyte tissue. Collectively, these findings reveal a novel critical role of ATF6 in regulating ER stress-mediated apoptosis in chondrocyte differentiation and the molecular mechanisms involved.

Different Roles of GRP78 on Cell Proliferation and Apoptosis in Cartilage Development.

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.

Study on the effect of IRE1α on cell growth and apoptosis via modulation PLK1 in ER stress response

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). Failure to adapt to ER stress causes the UPR to trigger apoptosis. Inositol-requiring enzyme-1α (IRE1α), as one of three unfolded protein sensors in UPR signaling pathways, senses ER unfolded proteins through an ER lumenal domain that becomes oligomerized during ER stress. It is known to be important for ER stress-mediated apoptosis and cell growth, but the exact molecular mechanism underlying these processes remains unexplored. In this study, we report that knockdown of IRE1α by an siRNA silencing approach enhanced, whereas its overexpression inhibited, cell proliferation in Hepatoma cells. Besides, overexpression of IRE1α induced, while its repression inhibited, ER stress-mediated apoptosis in Hepatomas cells. Furthermore, we found that overexpressed IRE1α can down-regulate Polo-like kinase 1(PLK1) from mRNA and protein two levels. IRE1α-mediated induction of apoptosis and inhibition of proliferation in response to ER stress is through downregulation PLK1, an early trigger for G2/M transition known to be participated in regulating cell proliferation and cell apoptosis. Collectively, these findings reveal a novel critical role of IRE1α in ER stress-mediated apoptosis and the molecular mechanisms involved. IRE1α may be a useful molecular target for the development of novel predictive and therapeutic strategies in cancer.

Transmission of ER stress response by ATF6 promotes endochondral bone growth

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.

XBP1S, a BMP2‐inducible transcription factor, accelerates endochondral bone growth by activating GEP growth factor

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.

Regulation of chondrocyte differentiation by IRE1α depends on its enzymatic activity

Bone morphogenetic protein 2(BMP2) is known to activate unfolded protein response (UPR) signal molecules in chondrogenesis. Inositol-requiring enzyme-1α (IRE1α),as one of three unfolded protein sensors in UPR signaling pathways, can be activated during ER stress. However, the influence on IRE1α in chondrocyte differentiation has not yet been elucidated. Here we present evidence demonstrating that overexpression of IRE1α inhibits chondrocyte differentiation, as revealed by reduced expression of collagen II (ColII), Sox9, collagen X (ColX), matrix metalloproteinase 13 (MMP-13), Indian hedgehog (IHH), Runx2 and enhanced expression of parathyroid hormone-related peptide (PTHrP). Furthermore, IRE1α-mediated inhibition of chondrogenesis depends on its enzymatic activity, since its point mutant lacking enzymatic activity completely loses this activity. The RNase and Kinase domains of IRE1α C-terminal are necessary for its full enzymatic activity and inhibition of chondrocyte differentiation. Mechanism studies demonstrate that granulin-epithelin precursor(GEP), a growth factor known to stimulate chondrogenesis, induced IRE1α expression in chondrogenesis. The expression of IRE1α is depended on GEP signaling, and IRE1α expression is hardly detectable in GEP(-/-) embryos. In addition, IRE1α inhibits GEP-mediated chondrocyte differentiation as a negative regulator. Altered expression of IRE1α in chondrocyte hypertrophy was accompanied by altered levels of IHH and PTHrP. Collectively, IRE1α may be a novel regulator of chondrocyte differentiation by 1) inhibition GEP-mediated chondrocyte differentiation as a negative regulator; 2) promoting IHH/PTHrP signaling.