Yun Hyun Huh

REGULATION OF TYPE II COLLAGEN EXPRESSION BY HISTONE DEACETYLASE IN ARTICULAR CHONDROCYTES

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.

Hypoxia-inducible factor-2α is an essential catabolic regulator of inflammatory rheumatoid arthritis.

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.

Swiprosin-1 modulates actin dynamics by regulating the F-actin accessibility to cofilin

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.

Low-density lipoprotein receptor–related protein 5 governs Wnt-mediated osteoarthritic cartilage destruction

IntroductionWnt ligands bind to low-density lipoprotein receptor–related protein (LRP) 5 or 6, triggering a cascade of downstream events that include β-catenin signaling. Here we explored the roles of LRP5 in interleukin 1β (IL-1β)- or Wnt-mediated osteoarthritic (OA) cartilage destruction in mice.MethodsThe expression levels of LRP5, type II collagen, and catabolic factors were determined in mouse articular chondrocytes, human OA cartilage, and mouse experimental OA cartilage. Experimental OA in wild-type, Lrp5 total knockout (Lrp5-/-) and chondrocyte-specific knockout (Lrp5fl/fl;Col2a1-cre) mice was caused by aging, destabilization of the medial meniscus (DMM), or intra-articular injection of collagenase. The role of LRP5 was confirmed in vitro by small interfering RNA–mediated knockdown of Lrp5 or in Lrp5-/- cells treated with IL-1β or Wnt proteins.ResultsIL-1β treatment increased the expression of LRP5 (but not LRP6) via JNK and NF-κB signaling. LRP5 was upregulated in human and mouse OA cartilage, and Lrp5 deficiency in mice inhibited cartilage destruction. Treatment with IL-1β or Wnt decreased the level of Col2a1 and increased those of Mmp3 or Mmp13, whereas Lrp5 knockdown ameliorated these effects. In addition, we found that the functions of LRP5 in arthritic cartilage were subject to transcriptional activation by β-catenin. Moreover, Lrp5-/- and Lrp5fl/fl;Col2a1-cre mice exhibited decreased cartilage destruction (and related changes in gene expression) in response to experimental OA.ConclusionsOur findings indicate that LRP5 (but not LRP6) plays an essential role in Wnt/β-catenin-signaling-mediated OA cartilage destruction in part by regulating the expression levels of type II collagen, MMP3, and MMP13.

Crosstalk between FLS and chondrocytes is regulated by HIF-2α-mediated cytokines in arthritis.

Rheumatoid arthritis (RA) and osteoarthritis (OA), two common types of arthritis, affect the joints mainly by targeting the synovium and cartilage. Increasing evidence indicates that a significant network connects synovitis and cartilage destruction during the progression of arthritis. We recently demonstrated that hypoxia-inducible factor (HIF)-2α causes RA and OA by regulating the expression of catabolic factors in fibroblast-like synoviocytes (FLS) or chondrocytes. To address the reciprocal influences of HIF-2α on FLS and chondrocytes, we applied an in vitro co-culture system using a transwell apparatus. When co-cultured with HIF-2α-overexpressing chondrocytes, FLS exhibited increased expression of matrix metalloproteinases and inflammatory mediators, similar to the effects induced by tumor-necrosis factor (TNF)-α treatment of FLS. Moreover, chondrocytes co-cultured with HIF-2α-overexpressing FLS exhibited upregulation of Mmp3 and Mmp13, which is similar to the effects induced by interleukin (IL)-6 treatment of chondrocytes. We confirmed these differential HIF-2α-induced effects via distinct secretory mediators using Il6-knockout cells and a TNF-α-blocking antibody. The FLS-co-culture-induced gene expression changes in chondrocytes were significantly abrogated by IL-6 deficiency, whereas TNF-α neutralization blocked the alterations in gene expression associated with co-culture of FLS with chondrocytes. Our results further suggested that the observed changes might reflect the HIF-2α-induced upregulation of specific receptors for TNF-α (in FLS) and IL-6 (in chondrocytes). This study broadens our understanding of the possible regulatory mechanisms underlying the crosstalk between the synovium and cartilage in the presence of HIF-2α, and may suggest potential new anti-arthritis therapies.

HIF-2α-induced chemokines stimulate motility of fibroblast-like synoviocytes and chondrocytes into the cartilage-pannus interface in experimental rheumatoid arthritis mouse models

IntroductionPannus formation and resulting cartilage destruction during rheumatoid arthritis (RA) depends on the migration of synoviocytes to cartilage tissue. Here, we focused on the role of hypoxia-inducible factor (HIF)-2α-induced chemokines by chondrocytes in the regulation of fibroblast-like synoviocyte (FLS) migration into the cartilage-pannus interface and cartilage erosion.MethodsCollagen-induced arthritis (CIA), K/BxN serum transfer, and tumor necrosis factor-α transgenic mice were used as experimental RA models. Expression patterns of HIF-2α and chemokines were determined via immunostaining, Western blotting and RT-PCR. FLS motility was evaluated using transwell migration and invasion assays. The specific role of HIF-2α was determined via local deletion of HIF-2α in joint tissues or using conditional knockout (KO) mice. Cartilage destruction, synovitis and pannus formation were assessed via histological analysis.ResultsHIF-2α and various chemokines were markedly upregulated in degenerating cartilage and pannus of RA joints. HIF-2α induced chemokine expression by chondrocytes in both primary culture and cartilage tissue. HIF-2α -induced chemokines by chondrocytes regulated the migration and invasion of FLS. Local deletion of HIF-2α in joint tissues inhibited pannus formation adjacent to cartilage tissue and cartilage destruction caused by K/BxN serum transfer. Furthermore, conditional knockout of HIF-2α in cartilage blocked pannus formation in adjacent cartilage but not bone tissue, along with inhibition of cartilage erosion caused by K/BxN serum transfer.ConclusionOur findings suggest that chemokines induced by IL-1β or HIF-2α in chondrocytes regulate pannus expansion by stimulating FLS migration and invasion, leading to cartilage erosion during RA pathogenesis.

SPIN90 depletion and Microtubule Acetylation Mediate Stromal Fibroblast Activation in Breast Cancer Progression

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. Spin90 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. Spin90 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. Cancer Res; 77(17); 4710-22. ©2017 AACR.

NAMPT Is an Essential Regulator of RA-Mediated Periodontal Inflammation:

Recent studies have indicated a potential correlation between rheumatoid arthritis (RA) and periodontal inflammation. We undertook this study to verify whether RA mediates periodontitis-like phenotypes in experimental mouse models of RA and to explore the role of nicotinamide phosphoribosyltransferase (NAMPT) in periodontal inflammation during RA pathogenesis. Periodontal inflammation and alveolar bone loss have been reported in mice with collagen-induced arthritis (CIA) and in genetically modified tumor necrosis factor–α (TNF-α) transgenic (TG) mouse models. Among the adipokines examined in our study, NAMPT expression was markedly upregulated in the periodontal ligament (PDL) tissues in RA mouse models and in human PDL cells stimulated by the proinflammatory cytokines, interleukin (IL) 1β and TNF-α. When NAMPT was overexpressed with the Nampt-synthesizing adenovirus vector (Ad-Nampt), the PDL cells exhibited an increased expression of cytokines (IL6), chemokines (IL8 and chemokine [C-C motif] ligand 5 [CCL5]), inflammatory mediators (cyclooxygenase 2 [COX-2]), and matrix-degrading enzymes (matrix metalloproteinase [MMP] 1 and MMP3). Inhibition of NAMPT by the intracellular NAMPT (iNAMPT) inhibitor, FK866, or by the sirtuin inhibitor, nicotinamide, in PDL cells led to inhibition of the IL1β or Ad-Nampt–induced upregulation of catabolic factors, whereas treatment with recombinant NAMPT protein or blockade of extracellular NAMPT (eNAMPT) with blocking antibody did not. Moreover, NAMPT inhibition by the intraperitoneal or intragingival injection of FK866 in CIA mice inhibited periodontal tissue damage, under conditions of RA. Thus, our results verified the co-occurrence of RA and periodontal inflammation using experimental mouse models of RA, suggesting that iNAMPT in PDL cells plays a pivotal role in the pathogenesis of RA-mediated periodontal inflammation by regulating the expression levels of catabolic genes, such as IL6, IL8, CCL5, COX-2, MMP1, and MMP3.

SPIN90 Modulates Long-Term Depression and Behavioral Flexibility in the Hippocampus

The importance of actin-binding proteins (ABPs) in the regulation of synapse morphology and plasticity has been well established. SH3 protein interacting with Nck, 90 kDa (SPIN90), an Nck-interacting protein highly expressed in synapses, is essential for actin remodeling and dendritic spine morphology. Synaptic targeting of SPIN90 to spine heads or dendritic shafts depends on its phosphorylation state, leading to blockage of cofilin-mediated actin depolymerization and spine shrinkage. However, the physiological role of SPIN90 in long-term plasticity, learning and memory are largely unknown. In this study, we demonstrate that Spin90-knockout (KO) mice exhibit substantial deficits in synaptic plasticity and behavioral flexibility. We found that loss of SPIN90 disrupted dendritic spine density in CA1 neurons of the hippocampus and significantly impaired long-term depression (LTD), leaving basal synaptic transmission and long-term potentiation (LTP) intact. These impairments were due in part to deficits in AMPA receptor endocytosis and its pre-requisites, GluA1 dephosphorylation and postsynaptic density (PSD) 95 phosphorylation, but also by an intrinsic activation of Akt-GSK3β signaling as a result of Spin90-KO. In accordance with these defects, mice lacking SPIN90 were found to carry significant deficits in object-recognition and behavioral flexibility, while learning ability was largely unaffected. Collectively, these findings demonstrate a novel modulatory role for SPIN90 in hippocampal LTD and behavioral flexibility.

NAMPT enzyme activity regulates catabolic gene expression in gingival fibroblasts during periodontitis

Periodontal disease is one of the most prevalent chronic disorders worldwide. It is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss. Here, we focused on the role of adipokines, which are locally expressed by periodontal tissues, in the regulation of catabolic gene expression leading to periodontal inflammation. The expression of the nicotinamide phosphoribosyltransferase (NAMPT) adipokine was dramatically increased in inflamed human and mouse gingival tissues. NAMPT expression was also increased in lipopolysaccharide- and proinflammatory cytokine-stimulated primary cultured human gingival fibroblasts (GF). Adenovirus-mediated NAMPT (Ad-Nampt) overexpression upregulated the expression and activity of COX-2, MMP1 and MMP3 in human GF. The upregulation of IL-1β- or Ad-Nampt-induced catabolic factors was significantly abrogated by the intracellular NAMPT (iNAMPT) inhibitor, FK866 or by the sirtuin (SIRT) inhibitor, nicotinamide (NIC). Recombinant NAMPT protein or extracellular NAMPT (eNAMPT) inhibition using a blocking antibody did not alter NAMPT target gene expression levels. Moreover, intragingival Ad-Nampt injection mediated periodontitis-like phenotypes including alveolar bone loss in mice. SIRT2, a part of the SIRT family, was positively associated with NAMPT actions in human GF. Furthermore, in vivo inhibition of the NAMPT-NAD+-SIRT axis by NIC injection in mice ameliorated the periodontal inflammation and alveolar bone erosion caused by intragingival injection of Ad-Nampt. Our findings indicate that NAMPT is highly upregulated in human GF, while its enzymatic activity acts as a crucial mediator of periodontal inflammation and alveolar bone destruction via regulation of COX-2, MMP1, and MMP3 levels.