Upon cytokine treatment, members of the signal transducers and activators of transcription (STAT) family of proteins are phosphorylated on tyrosine and serine sites within the carboxyl-terminal region in cells. We show that in response to cytokine treatment, Stat3 is also acetylated on a single lysine residue, Lys685. Histone acetyltransferase p300-mediated Stat3 acetylation on Lys685 was reversible by type I histone deacetylase (HDAC). Use of a prostate cancer cell line (PC3) that lacks Stat3 and PC3 cells expressing wild-type Stat3 or a Stat3 mutant containing a Lys685-to-Arg substitution revealed that Lys685 acetylation was critical for Stat3 to form stable dimers required for cytokine-stimulated DNA binding and transcriptional regulation, to enhance transcription of cell growth-related genes, and to promote cell cycle progression in response to treatment with oncostatin M.

Cytokine-activated receptors initiate intracellular signaling by recruiting protein kinases that phosphorylate the receptors on tyrosine residues, thus enabling docking of SH2 domain-bearing activating factors. Here we report that in response to type 1 interferon (IFNalpha), IFNalpha receptors recruit cytoplasmic CREB-binding protein (CBP). By binding to IFNalphaR2 within the region where two adjacent proline boxes bear phospho-Ser364 and phospho-Ser384, CBP acetylates IFNalphaR2 on Lys399, which in turn serves as the docking site for interferon regulatory factor 9 (IRF9). IRF9 interacts with the acetyl-Lys399 motif by means of its IRF homology2 (IH2) domain, leading to formation of the ISGF3 complex that includes IRF9, STAT1, and STAT2. All three components are acetylated by CBP. Remarkably, acetylation within the DNA-binding domain (DBD) of both IRF9 and STAT2 is critical for the ISGF3 complex activation and its associated antiviral gene regulation. These results have significant implications concerning the central role of acetylation in cytokine receptor signal transduction.

Mechanical stress plays an essential role in tissue development and remodeling. In this study, we determined the role of microRNA in chondrocyte mechanotransduction. Using microarray, we identified miR-365 as a mechanoresponsive microRNA in parallel to mechanical induction of Indian hedgehog (Ihh) in primary chicken chondrocytes cultured in 3-dimensional collagen scaffoldings under cyclic loading (1 Hz, 5% elongation). Interestingly, expression of miR-365 is elevated in the prehypertrophic zone of the growth plate, coinciding with the Ihh expression region in vivo. MiR-365 significantly stimulates chondrocyte proliferation and differentiation. MiR-365 increases expression of Ihh and the hypertrophic marker type X collagen, whereas anti-miR-365 inhibits the expression of these genes. We identified histone deacetylase 4 (HDAC4), an inhibitor of chondrocyte hypertrophy, as a target of miR-365. MiR-365 inhibits both endogenous HDAC4 protein levels as well as the activity of a reporter gene bearing the 3'-untranslated region of HDAC4 mRNA. Conversely, inhibition of endogenous miR-365 relieves the repression of HDAC4. Mutation of the miR-365 binding site in HDAC4 mRNA abolishes miR-365-mediated repression of the reporter gene activity. Overexpression of HDAC4 reverses miR-365 stimulation of chondrocyte differentiation markers including Ihh, Col X, and Runx2. Moreover, inhibition of miR-365 abolishes mechanical stimulation of chondrocyte differentiation. Taken together, miR-365 is the first identified mechanically responsive microRNA that regulates chondrocyte differentiation via directly targeting HDAC4.

The receptor tyrosine kinases (RTKs) RET, MET, and RON all carry the Met p؉1loop 3Thr point mutation (i.e., 2B mutation), leading to the formation of tumors with high metastatic potential. Utilizing a novel antibody array, we identified constitutive phosphorylation of STAT3 in cells expressing the 2B mutation but not wild-type RET. MET or RON with the 2B mutation also constitutively phosphorylated STAT3. Members of the EPH, the only group of wild-type RTK that carry Thr p؉1loop residue, are often expressed unexpectedly in different types of cancers. Ectopic expression of wild-type but not Thr p؉1loop 3Met substituted EPH family members constitutively phosphorylated STAT3. In both RTK Metp؉1loop with 2B mutation and wild-type EPH members the Thr p؉1loop residue is required for constitutive kinase autophosphorylation and STAT3 recruitment. In multiple endocrine neoplasia 2B (MEN-2B) patients expressing RET M918T , nuclear enrichment of STAT3 and elevated expression of CXCR4 was detected in metastatic thyroid C-cell carcinoma in the liver. In breast adenocarcinoma cell lines expressing multiple EPH members, STAT3 constitutively bound to the promoters of MUC1, MUC4, and MUC5B genes. Inhibiting STAT3 expression resulted in reduced expression of these metastasis-related genes and inhibited mobility. These findings provide insight into Thr p؉1loop residue in RTK autophosphorylation and constitutive activation of STAT3 in metastatic cancer cells.Growth factor receptors are transmembrane protein tyrosine kinases playing critical roles in biological processes, including proliferation, survival, and migration. The core region of the kinase domain, comprising the catalytic loop, the activation loop, and the pϩ1 loop, is well conserved in receptor tyrosine kinases (RTKs) of different species. RTKs with different types of mutations within these three loops have been associated with developmental disorders and cancer. The point mutation (ATG3ACG), resulting in replacement of methionine with threonine within the pϩ1 loop, is associated with aggressive tumors. RET is an RTK that can activate a variety of signaling pathways, including the RAS/ERK, PI3K/AKT, and phospholipase C␥ pathways and plays an important role in neuron survival or differentiation (11). RET with a Met pϩ1loop 3Thr substitution (RET M918T ) is associated with the multiple endocrine neoplasia 2B type (MEN-2B) syndrome; this substitution is defined as the 2B mutation (11). In MEN-2B patients, the tumors derived from thyroid C cells are often more aggressive than C-cell tumors that develop in MEN-2A patients who carry mutations in the extracellular domain of RET (11,22). Similarly, the 2B mutation in HGF receptor MET (Met M1268T ) has been identified in metastatic renal carcinomas (10,24). Introduction of the 2B mutation in other RTKs, such as RON and epidermal growth factor receptor (EGFR), caused transformation of NIH 3T3 cells with high metastatic potential (20,23). Although the 2B mutation enhanced kinase activity and such a mutation has been suspected as a g...

Mechanical stress plays an important role in the initiation and progression of osteoarthritis. Studies show that excessive mechanical stress can directly damage the cartilage extracellular matrix and shift the balance in chondrocytes to favor catabolic activity over anabolism. However, the underlying mechanism remains unknown. MicroRNAs (miRNAs) are emerging as important regulators in osteoarthritis pathogenesis. We have found that mechanical loading up-regulated microRNA miR-365 in growth plate chondrocytes, which promotes chondrocyte differentiation. Here, we explored the role of the mechanical responsive microRNA miR-365 in pathogenesis of osteoarthritis (OA). We found that miR-365 was up-regulated by cyclic loading and IL-1β stimulation in articular chondrocytes through a mechanism that involved the transcription factor NF-κB. miR-365 expressed significant higher level in rat anterior cruciate ligament (ACL) surgery induced OA cartilage as well as human OA cartilage from primary OA patients and traumatic OA Patients. Overexpression of miR-365 in chondrocytes increases gene expression of matrix degrading enzyme matrix metallopeptidase 13 (MMP13) and collagen type X (Col X). The increase in miR-365 expression in OA cartilage and in response to IL-1 may contribute to the abnormal gene expression pattern characteristic of OA. Inhibition of miR-365 down-regulated IL-1β induced MMP13 and Col X gene expression. We further showed histone deacetylase 4 (HDAC4) is a direct target of miR-365, which mediates mechanical stress and inflammation in OA pathogenesis. Thus, miR-365 is a critical regulator of mechanical stress and pro-inflammatory responses, which contributes cartilage catabolism. Manipulation of the expression of miR-365 in articular chondrocytes by miR-365 inhibitor may be a potent therapeutic target for the prevention and treatment of osteoarthritis.

Cytokine-activated receptors undergo extracellular domain dimerization, which is necessary to activate intracellular signaling pathways. Here, we report that in prolactin (PRL)-treated cells, PRL receptor (PRLR) undergoes cytoplasmic loop dimerization that is acetylation-dependent. PRLR-recruited CREB-binding protein (CBP) acetylates multiple lysine sites randomly distributed along the cytoplasmic loop of PRLR. Two PRLR monomers appear to interact with each other at multiple parts from the membrane-proximal region to the membrane-distal region, relying on the coordination among multiple lysine sites neutralized via acetylation. Cytoplasmic loop-dimerized PRLR activates STAT5, which is also acetylated by CBP and undergoes acetylation-dependent dimerization. PRLR dimerization and subsequent signaling are enhanced by treating the cells with deacetylase sirtuin (SIRT) inhibitor nicotinamide or histone deacetylase (HDAC) inhibitor trichostatin A but inhibited by expressing exogenous deacetylase SIRT2 or HDAC6. Our results suggest that acetylation and deacetylation provide the rheostatlike regulation for the cytokine receptor PRLR in its cytoplasmic loop dimerization and subsequent STAT5 activation.acetylation | CREB-binding protein | dimerization | prolactin receptor | STAT5

Ankyrin repeat and SOCS box (ASB) family members have a C-terminal SOCS box and an N-terminal ankyrin-related sequence of variable repeats belonging to the SOCS superfamily. While SH2-domain-bearing SOCS proteins are mainly involved in the negative feedback regulation of the protein tyrosine kinase-STAT pathway in response to a variety of cytokines, the roles of ASB family members remain largely unknown. To investigate ASB functions, we screened for ASB3-interacting factors by using antibody array technology and identified tumor necrosis factor receptor II (TNF-R2) as an ASB3 binding target. ASB3 expression and activities are required for (i) TNF-R2 ubiquitination both in vivo and in vitro, (ii) TNF-R2 proteolysis via the proteasome pathway, and (iii) the inhibition of TNF-R2-mediated Jun N-terminal protein kinase (JNK) activation. While the ankyrin repeats of ASB3 interact with the C-terminal 37 amino acids of TNF-R2, the SOCS box of ASB3 is responsible for recruiting the E3 ubiquitin ligase adaptors Elongins-B/C, leading to TNF-R2 ubiquitination on multiple lysine residues within its C-terminal region. Downregulation of ASB3 expression by a small interfering RNA inhibited TNF-R2 degradation and potentiated TNF-R2-mediated cytotoxicity. The data presented here implicate ASB3 as a negative regulator of TNF-R2-mediated cellular responses to TNF-␣ by direct targeting of TNF-R2 for ubiquitination and proteasome-mediated degradation.The suppressor of cytokine signaling (SOCS) box has been identified as the functional domain of a diverse family of protein adapters, which include SOCS, ASB, WD-40 repeat and SOCS box (WSB), SPRY domain and SOCS box (SSB), and Ras-related protein Rab. The SOCS box is an approximately 40-to 60-amino-acid motif, and has a predicted helical structure (12). Among the eight SH2-domain-containing SOCS family members, SOCS-1 and SOCS-3 have been most extensively studied and have been shown to act in a classic negative feedback loop to inhibit JAK-STAT signal transduction. In SOCS-1 and SOCS-3, while the SH2 domains are responsible for recognizing phospho-tyrosine motifs of the substrates, as is the case for JAK (24,29,34), the SOCS box was found to associate with Elongins-B/C, two proteins that form an E3 ubiquitin ligase complex with Cullin-2 and Rbx-1 (14, 35). This raises the possibility that SOCS proteins may inhibit signaling by functioning as adaptors for an E3 ubiquitin ligase complex, which could mediate the ubiquitination of SOCS binding partners. More definitive evidence of SOCS box recruitment or an E3 ligase has come from studies of the von-Hippel-Lindau (VHL) protein. A structural analysis demonstrated a direct interaction between the VHL protein and the E3 ligase component Elongin-C (32). VHL binds to Elongin-C through the VHL␣ domain, which is structurally and functionally homologous to the SOCS box (32). Numerous tumors are caused by mutations within the VHL␣ domain. These mutations prevent Elongin-C recruitment to the VHL␣ domain, which results in the loss of its activity in i...

SummaryPrimary osteoarthritis (OA) is associated with aging, while post‐traumatic OA (PTOA) is associated with mechanical injury and inflammation. It is not clear whether the two types of osteoarthritis share common mechanisms. We found that miR‐146a, a microRNA‐associated with inflammation, is activated by cyclic load in the physiological range but suppressed by mechanical overload in human articular chondrocytes. Furthermore, miR‐146a expression is decreased in the OA lesions of human articular cartilage. To understand the role of miR‐146a in osteoarthritis, we systemically characterized mice in which miR‐146a is either deficient in whole body or overexpressed in chondrogenic cells specifically. miR‐146a‐deficient mice develop early onset of OA characterized by cartilage degeneration, synovitis, and osteophytes. Conversely, miR‐146a chondrogenic overexpressing mice are resistant to aging‐associated OA. Loss of miR‐146a exacerbates articular cartilage degeneration during PTOA, while chondrogenic overexpression of miR‐146a inhibits PTOA. Thus, miR‐146a inhibits both OA and PTOA in mice, suggesting a common protective mechanism initiated by miR‐146a. miR‐146a suppresses IL‐1β of catabolic factors, and we provide evidence that miR‐146a directly inhibits Notch1 expression. Therefore, such inhibition of Notch1 may explain suppression of inflammatory mediators by miR‐146a. Chondrogenic overexpression of miR‐146a or intra‐articular administration of a Notch1 inhibitor alleviates IL‐1β‐induced catabolism and rescues joint degeneration in miR‐146a‐deficient mice, suggesting that miR‐146a is sufficient to protect OA pathogenesis by inhibiting Notch signaling in the joint. Thus, miR‐146a may be used to counter both aging‐associated OA and mechanical injury‐/inflammation‐induced PTOA.