Hypoxia-inducible factor-2α (HIF-2α) is sufficient to cause experimental rheumatoid arthritis and acts to regulate the functions of fibroblast-like cells from tissue surrounding joints, independent of HIF-1α.

Pathological bone loss is caused by an imbalance between bone formation and resorption. The bone microenvironments are hypoxic, and hypoxia-inducible factor (HIF) is known to play notable roles in bone remodeling. However, the relevant functions of HIF-2α are not well understood. Here, we have shown that HIF-2α deficiency in mice enhances bone mass through its effects on the differentiation of osteoblasts and osteoclasts. In vitro analyses revealed that HIF-2α inhibits osteoblast differentiation by targeting Twist2 and stimulates RANKL-induced osteoclastogenesis via regulation of Traf6 . In addition, HIF-2α appears to contribute to the crosstalk between osteoblasts and osteoclasts by directly targeting RANKL in osteoprogenitor cells. Experiments performed with osteoblast- and osteoclast-specific conditional knockout mice supported a role of HIF-2α in this crosstalk. HIF-2α deficiency alleviated ovariectomy-induced bone loss in mice, and specific inhibition of HIF-2α with ZINC04179524 significantly blocked RANKL-mediated osteoclastogenesis. Collectively, our results suggest that HIF-2α functions as a catabolic regulator in bone remodeling, which is critical for the maintenance of bone homeostasis.

In response to brain injury, microglia rapidly extend processes that isolate lesion sites and protect the brain from further injury. Here we report that microglia carrying a pathogenic mutation in the Parkinson's disease (PD)-associated gene, G2019S-LRRK2 (GS-Tg microglia), show retarded ADP-induced motility and delayed isolation of injury, compared with non-Tg microglia. Conversely, LRRK2 knockdown microglia are highly motile compared with control cells. In our functional assays, LRRK2 binds to focal adhesion kinase (FAK) and phosphorylates its Thr–X–Arg/Lys (TXR/K) motif(s), eventually attenuating FAK activity marked by decreased pY397 phosphorylation (pY397). GS-LRRK2 decreases the levels of pY397 in the brain, microglia and HEK cells. In addition, treatment with an inhibitor of LRRK2 kinase restores pY397 levels, decreased pTXR levels and rescued motility of GS-Tg microglia. These results collectively suggest that G2019S mutation of LRRK2 may contribute to the development of PD by inhibiting microglial response to brain injury.

Histone deacetylase (HDAC) regulates various cellular processes by modulating gene expression. Here, we investigated the role of HDAC in the expression of type II collagen, a marker of differentiated chondrocytes. We found that HDAC activity in primary articular chondrocytes decreases during dedifferentiation induced by serial monolayer culture and that the activity recovered during redifferentiation induced by three-dimensional culture in a cell pellet. Inhibition of HDAC with trichostatin A or PXD101 was sufficient to block type II collagen expression in primary culture chondrocytes. HDAC inhibition also blocked the redifferentiation of dedifferentiated chondrocytes by suppressing the synthesis and accumulation of type II collagen. HDAC inhibition promoted the expression of Wnt-5a, which is known to inhibit type II collagen expression, and knockdown of Wnt-5a blocked the ability of HDAC inhibitors to suppress type II collagen expression. In addition, the induction of Wnt-5a expression by HDAC inhibitors was associated with acetylation of the Wnt-5a promoter. Taken together, our results suggest that HDAC promotes type II collagen expression by suppressing the transcription of Wnt-5a.Differentiation of uncommitted mesenchymal cells into chondrocytes is initiated by the proliferation and recruitment of chondrogenic mesenchymal cells into condensations. These precartilage condensations develop into cartilage nodules containing differentiated chondrocytes. Chondrogenesis can be mimicked by micromass culture of mesenchymal cells in vitro. Differentiated chondrocytes are characterized by the ability to synthesize cartilage-specific extracellular matrix molecules including type II collagen and sulfated proteoglycans, which are necessary for normal cartilage development (1, 2). The activity of cartilage-specific matrix molecule synthesis by the differentiated chondrocytes is unstable and rapidly lost in response to certain environmental changes. For instance, proinflammatory cytokines such as interleukin (IL) 2 -1␤ cause the loss of differentiated chondrocyte phenotypes (dedifferentiation) by halting the synthesis of cartilage-specific matrix molecules (3, 4). Chondrocyte dedifferentiation is also induced by serial monolayer culture and is accompanied by a gradual shift in the expression of type II collagen and aggrecan to type I and III collagen and versican, respectively (5, 6). Dedifferentiated chondrocytes redifferentiate in three-dimensional culture in an alginate gel or cell pellet (6, 7). Although maintenance of a differentiated chondrocyte phenotype is important for cartilage homeostasis, the detailed mechanisms of the differentiation and dedifferentiation of chondrocytes remain largely unknown. Because chondrocyte differentiation/dedifferentiation is regulated by various soluble factors including growth factors and cytokines, we hypothesized that histone deacetylase (HDAC) may regulate the differentiation status of chondrocytes. HDAC modulates the growth and differentiation of various cell types by governing...

Membrane protrusions, like lamellipodia, and cell movement are dependent on actin dynamics, which are regulated by a variety of actin-binding proteins acting cooperatively to reorganize actin filaments. Here, we provide evidence that Swiprosin-1, a newly identified actin-binding protein, modulates lamellipodial dynamics by regulating the accessibility of F-actin to cofilin. Overexpression of Swiprosin-1 increased lamellipodia formation in B16F10 melanoma cells, whereas knockdown of Swiprosin-1 inhibited EGF-induced lamellipodia formation, and led to a loss of actin stress fibers at the leading edges of cells but not in the cell cortex. Swiprosin-1 strongly facilitated the formation of entangled or clustered F-actin, which remodeled the structural organization of actin filaments making them inaccessible to cofilin. EGF-induced phosphorylation of Swiprosin-1 at Ser183, a phosphorylation site newly identified using mass spectrometry, effectively inhibited clustering of actin filaments and permitted cofilin access to F-actin, resulting in actin depolymerization. Cells overexpressing a Swiprosin-1 phosphorylation-mimicking mutant or a phosphorylation-deficient mutant exhibited irregular membrane dynamics during the protrusion and retraction cycles of lamellipodia. Taken together, these findings suggest that dynamic exchange of Swiprosin-1 phosphorylation and dephosphorylation is a novel mechanism that regulates actin dynamics by modulating the pattern of cofilin activity at the leading edges of cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-013-1447-5) contains supplementary material, which is available to authorized users.

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Ectopic expression of Swiprosin-1, an actin-binding protein (also known as EF hand domain containing 2; EFHD2), enhanced motile protrusions associated with actin, such as lamellipodia and membrane ruffles. Swiprosin-1 levels were increased in various human cancer tissues, particularly at highly invasive stages of malignant melanoma. Expression of Swiprosin-1 was correlated with that of epidermal growth factor receptor (EGFR) and induced by EGF. In a mouse metastasis model, Swiprosin-1 overexpression induced pulmonary metastasis whereas its knockdown led to marked inhibition of metastasis of highly invasive melanoma cells. Swiprosin-1 at the lamellipodia and membrane ruffles controlled the direction of cell protrusion and enhanced migration velocity through activating the Rho family of small GTPases, including Rac1, Cdc42 and RhoA. Our collective findings support the potential utility of Swiprosin-1 as a therapeutic target to prevent cancer invasion and metastasis.

Biomechanical remodeling of stroma by cancer-associated fibroblasts (CAF) in early stages of cancer is critical for cancer progression, and mechanical cues such as extracellular matrix stiffness control cell differentiation and malignant progression. However, the mechanism by which CAF activation occurs in low stiffness stroma in early stages of cancer is unclear. Here, we investigated the molecular mechanism underlying CAF regulation by SPIN90 and microtubule acetylation under conditions of mechanically soft matrices corresponding to normal stromal rigidity. SPIN90 was downregulated in breast cancer stroma but not tumor, and this low stromal expression correlated with decreased survival in breast cancer patients. deficiency facilitated recruitment of mDia2 and APC complex to microtubules, resulting in increased microtubule acetylation. This increased acetylation promoted nuclear localization of YAP, which upregulated expression of myofibroblast marker genes on soft matrices. depletion enhanced tumor progression, and blockade of microtubule acetylation in CAF significantly inhibited tumor growth in mice. Together, our data demonstrate that loss of SPIN90-mediated microtubule acetylation is a key step in CAF activation in low stiffness stroma. Moreover, correlation among these factors in human breast cancer tissue supports the clinical relevance of SPIN90 and microtubule acetylation in tumor development. .