Hic-5 Protein Research Articles

Knockdown of mechanosensitive adaptor Hic-5 ameliorates post-traumatic osteoarthritis in rats through repression of MMP-13

Osteoarthritis (OA) is the most common joint disease associated with articular cartilage destruction. Matrix metalloproteinase-13 (MMP-13) has an essential role in OA pathogenesis by degradation of collagen II, a major component of articular cartilage. Hydrogen peroxide-inducible clone-5 (Hic-5; TGFB1I1), a transforming growth factor-β-inducible mechanosensor, has previously been reported to promote OA pathogenesis by upregulating MMP-13 expression in mouse osteoarthritic lesions. In our current study, immunohistochemical analysis showed that Hic-5 protein expression was increased in human OA cartilage compared with normal cartilage. Functional experiments demonstrated that Hic-5 and MMP-13 expression was increased by mechanical stress, and mechanical stress-induced MMP-13 expression was suppressed by Hic-5 siRNA in human chondrocytes. Moreover, intracellular localization of Hic-5 shifted to the nucleus from focal adhesions in human chondrocytes subjected to mechanical stress, and nuclear Hic-5 increased MMP-13 gene expression. In vivo, intra-articular injection of Hic-5 siRNA decreased the Osteoarthritis Research Society International score and MMP-13 protein expression in articular cartilage of OA rats. Our findings suggest that Hic-5 regulates transcription of MMP-13 in human chondrocytes, and Hic-5 may be a novel therapeutic target for OA because OA progression was suppressed by intra-articular injection of Hic-5 siRNA in rats.

Increased expression of the transforming growth factor β-inducible gene HIC-5 in systemic sclerosis skin and fibroblasts: a novel antifibrotic therapeutic target.

SSc is a systemic fibrotic disease affecting skin, numerous internal organs and the microvasculature. The molecular pathogenesis of SSc tissue fibrosis has not been fully elucidated, although TGF-β1 plays a crucial role. The Hic-5 protein encoded by the TGF-β1-inducible HIC-5 gene participates in numerous TGF-β-mediated pathways, however, the role of Hic-5 in SSc fibrosis has not been investigated. The aim of this study was to examine HIC-5 involvement in SSc tissue fibrosis. Affected skin from three patients with diffuse SSc and dermal fibroblasts cultured from affected and non-affected SSc skin were examined for HIC-5 and COL1A1 gene expression. Real-time PCR, IF microscopy, western blotting and small interfering RNA-mediated HIC-5 were performed. HIC-5 and COL1A1 transcripts and Hic-5, type 1 collagen (COL1) and α-smooth muscle actin (α-SMA) protein levels were increased in clinically affected SSc skin compared with normal skin and in cultured dermal fibroblasts from affected SSc skin compared with non-affected skin fibroblasts from the same patients. HIC-5 knockdown caused a marked reduction of COL1 production in SSc dermal fibroblasts. HIC-5 expression is increased in affected SSc skin compared with skin from normal individuals. Affected SSc skin fibroblasts display increased HIC-5 and COL1A1 expression compared with non-affected skin fibroblasts from the same patients. Hic-5 protein was significantly increased in cultured SSc dermal fibroblasts. HIC-5 mRNA knockdown in SSc fibroblasts caused >50% reduction of COL1 production. Although these are preliminary results owing to the small number of skin samples studied, they indicate that Hic-5 plays a role in the profibrotic activation of SSc dermal fibroblasts and may represent a novel molecular target for antifibrotic therapy in SSc.

Role of extracellular matrix in breast cancer development: a brief update.

Evidence is increasing on the crucial role of the extracellular matrix (ECM) in breast cancer progression, invasion and metastasis with almost all mortality cases owing to metastasis. The epithelial-mesenchymal transition is the first signal of metastasis involving different transcription factors such as Snail, TWIST, and ZEB1. ECM remodeling is a major event promoting cancer invasion and metastasis; where matrix metalloproteinases (MMPs) such as MMP-2, -9, -11, and -14 play vital roles degrading the matrix proteins for cancer spread. The β-D mannuronic acid (MMP inhibitor) has anti-metastatic properties through inhibition of MMP-2, and -9 and could be a potential therapeutic agent. Besides the MMPs, the enzymes such as LOXL2, LOXL4, procollagen lysyl hydroxylase-2, and heparanase also regulate breast cancer progression. The important ECM proteins like integrins (b1-, b5-, and b6- integrins), ECM1 protein, and Hic-5 protein are also actively involved in breast cancer development. The stromal cells such as tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), and adipocytes also contribute in tumor development through different processes. The TAMs become proangiogenic through secretion of VEGF-A and building vessel network for nourishment and invasion of the tumor mass. The latest developments of ECM involvement in breast cancer progression has been discussed in this review and this study will help researchers in designing future work on breast cancer pathogenesis and developing therapy targeted to the ECM components.

Role of extracellular matrix in breast cancer development: a brief update

Evidence is increasing on the crucial role of the extracellular matrix (ECM) in breast cancer progression, invasion and metastasis with almost all mortality cases owing to metastasis. The epithelial-mesenchymal transition is the first signal of metastasis involving different transcription factors such as Snail, TWIST, and ZEB1. ECM remodeling is a major event promoting cancer invasion and metastasis; where matrix metalloproteinases (MMPs) such as MMP-2, -9, -11, and -14 play vital roles degrading the matrix proteins for cancer spread. The β-D mannuronic acid (MMP inhibitor) has anti-metastatic properties through inhibition of MMP-2, and -9 and could be a potential therapeutic agent. Besides the MMPs, the enzymes such as LOXL2, LOXL4, procollagen lysyl hydroxylase-2, and heparanase also regulate breast cancer progression. The important ECM proteins like integrins (b1-, b5-, and b6- integrins), ECM1 protein, and Hic-5 protein are also actively involved in breast cancer development. The stromal cells such as tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), and adipocytes also contribute in tumor development through different processes. The TAMs become proangiogenic through secretion of VEGF-A and building vessel network for nourishment and invasion of the tumor mass. The latest developments of ECM involvement in breast cancer progression has been discussed in this review and this study will help researchers in designing future work on breast cancer pathogenesis and developing therapy targeted to the ECM components.

Hic-5 Regulates Actin Cytoskeletal Reorganization and Expression of Fibrogenic Markers and Myocilin in Trabecular Meshwork Cells.

To explore the role of inducible focal adhesion (FA) protein Hic-5 in actin cytoskeletal reorganization, FA formation, fibrogenic activity, and expression of myocilin in trabecular meshwork (TM) cells. Using primary cultures of human TM (HTM) cells, the effects of various external factors on Hic-5 protein levels, as well as the effects of recombinant Hic-5 and Hic-5 small interfering RNA (siRNA) on actin cytoskeleton, FAs, myocilin, α-smooth muscle actin (αSMA), and collagen-1 were determined by immunofluorescence and immunoblot analyses. Hic-5 distributes discretely to the FAs in HTM cells and throughout the TM and Schlemm's canal of the human aqueous humor (AH) outflow pathway. Transforming growth factor-β2 (TGF-β2), endothelin-1, lysophosphatidic acid, hydrogen peroxide, and RhoA significantly increased Hic-5 protein levels in HTM cells in association with reorganization of actin cytoskeleton and FAs. While recombinant Hic-5 induced actin stress fibers, FAs, αv integrin redistribution to the FAs, increased levels of αSMA, collagen-1, and myocilin, Hic-5 siRNA suppressed most of these responses in HTM cells. Hic-5 siRNA also suppressed TGF-β2-induced fibrogenic activity and dexamethasone-induced myocilin expression in HTM cells. Taken together, these results reveal that Hic-5, whose levels were increased by various external factors implicated in elevated intraocular pressure, induces actin cytoskeletal reorganization, FAs, expression of fibrogenic markers, and myocilin in HTM cells. These characteristics of Hic-5 in TM cells indicate its importance in regulation of AH outflow through the TM in both normal and glaucomatous eyes.

Negative Regulation of NADPH Oxidase 4 by Hydrogen Peroxide-inducible Clone 5 (Hic-5) Protein

Hydrogen peroxide-inducible clone 5 (Hic-5) is a focal adhesion adaptor protein induced by the profibrotic cytokine TGF-β1. We have demonstrated previously that TGF-β1 induces myofibroblast differentiation and lung fibrosis by activation of the reactive oxygen species-generating enzyme NADPH oxidase 4 (Nox4). Here we investigated a potential role for Hic-5 in regulating Nox4, myofibroblast differentiation, and senescence. In normal human diploid fibroblasts, TGF-β1 induces Hic-5 expression in a delayed manner relative to the induction of Nox4 and myofibroblast differentiation. Hic-5 silencing induced constitutive Nox4 expression and enhanced TGF-β1-inducible Nox4 levels. The induction of constitutive Nox4 protein in Hic-5-silenced cells was independent of transcription and translation and controlled by the ubiquitin-proteasomal system. Hic-5 associates with the ubiquitin ligase Cbl-c and the ubiquitin-binding protein heat shock protein 27 (HSP27). The interaction of these proteins is required for the ubiquitination of Nox4 and for maintaining low basal levels of this reactive oxygen species-generating enzyme. Our model suggests that TGF-β1-induced Hic-5 functions as a negative feedback mechanism to limit myofibroblast differentiation and senescence by promoting the ubiquitin-proteasomal system-mediated degradation of Nox4. Together, these studies indicate that endogenous Hic-5 suppresses senescence and profibrotic activities of myofibroblasts by down-regulating Nox4 protein expression. Additionally, these are the first studies, to our knowledge, to demonstrate posttranslational regulation of Nox4.

Glomerular Expression of Hydrogen Peroxide-Inducible Clone-5 in Human and Rat Progressive Mesangial Proliferative Glomerulonephritis

Background/Aims: Hydrogen peroxide-inducible clone-5 (Hic-5) is a transforming growth factor-β₁ (TGF-β₁)- and hydrogen peroxide (H₂O₂)-inducible focal adhesion protein that may be necessary for maintaining the myofibroblastic phenotype in pathological scar formation. To investigate the involvement of Hic-5 in the pathogenesis of glomerulonephritis (GN), we examined the glomerular expression of Hic-5 in human and rat GN as well as the regulation of Hic-5 by TGF-β₁ in vitro. Methods and Results: Immunohistochemical analyses showed that the expression of Hic-5 was increased in mesangial cells (MCs) in human mesangial proliferative GN. Hic-5 expression was significantly correlated not only with the levels of α-smooth muscle actin (α-SMA) and TGF-β₁, the accumulation of extracellular matrix, and the number of glomerular cells, but also with the urinary protein level in patients with GN. Glomerular Hic-5 expression increased in parallel with α-SMA expression in a rat model of mesangial proliferative GN. Combined therapy with an angiotensin type I receptor blocker and an antioxidant in this model improved the histology and the expression of Hic-5 and α-SMA. TGF-β₁ upregulated Hic-5 and α-SMA protein levels in human cultured MCs. Conclusion: Our findings suggest that Hic-5 is involved in changes in the MC phenotype to produce abnormal extracellular matrix remodeling in GN.

Hic-5 as a regulator of endothelial cell morphology and connective tissue growth factor gene expression

The functional role of the LIM-domain protein Hic-5 was investigated in microvascular endothelial cells using a siRNA approach. Knock down of Hic-5 reduced endothelial cell spreading and impaired structural organization of the cells on basement membrane extracts. Furthermore, Hic-5 was involved in the regulation of the multifunctional protein connective tissue growth factor (CTGF, CCN2). Upon Hic-5 down-regulation, induction of CTGF by lysophosphatidic acid or colchicine was reduced. Inhibition of CTGF expression was even more pronounced in cells treated with transforming growth factor beta and inhibitors of histone deacetylases. Treatment of endothelial cells with Hic-5 siRNA reduced CTGF promoter activity. Mutation analyses of the promoter revealed transcription factors binding to the basic control element as part of the proposed Hic-5-modulated transcription complex. Further analyses showed down-regulation of Hic-5 protein upon overnight treatment with inhibitors of histone deacetylases. These data suggest that the reduced expression of Hic-5 may contribute to the anti-angiogenic effects of histone deacetylase inhibitors.

Focal Adhesion Kinase-Related Proline-Rich Tyrosine Kinase 2 and Focal Adhesion Kinase Are Co-Overexpressed in Early-Stage and Invasive ErbB-2-Positive Breast Cancer and Cooperate for Breast Cancer Cell Tumorigenesis and Invasiveness

Early cancer cell migration and invasion of neighboring tissues are mediated by multiple events, including activation of focal adhesion signaling. Key regulators include the focal adhesion kinase (FAK) and FAK-related proline-rich tyrosine kinase 2 (Pyk2), whose distinct functions in cancer progression remain unclear. Here, we compared Pyk2 and FAK expression in breast cancer and their effects on ErbB-2-induced tumorigenesis and the potential therapeutic utility of targeting Pyk2 compared with FAK in preclinical models of breast cancer. Pyk2 is overexpressed in tissues from early and advanced breast cancers and overexpressed with both FAK and epidermal growth factor receptor-2 (ErbB-2) in a subset of breast cancer cases. Down-regulation of Pyk2 in ErbB-2-positive, FAK-proficient, and FAK-deficient cells reduced cell proliferation, which correlated with reduced mitogen-activated protein kinase (MAPK) activity. In contrast, Pyk2 silencing had little impact on cell migration and invasion. In vivo, Pyk2 down-regulation reduced primary tumor growth induced by a metastatic variant of ErbB-2-positive MDA 231 breast cancer cells but had little effect on lung metastases in contrast to FAK down-regulation. Dual reduction of Pyk2 and FAK expression resulted in strong inhibition of both primary tumor growth and lung metastases. Together, these data support the cooperative function of Pyk2 and FAK in breast cancer progression and suggest that dual inhibition of FAK and Pyk2 is an efficient therapeutic approach for targeting invasive breast cancer.

Transforming Growth Factor-β1 Effects on Endothelial Monolayer Permeability Involve Focal Adhesion Kinase/Src

Transforming growth factor (TGF)-beta1 activity has been shown to increase vascular endothelial barrier permeability, which is believed to precede several pathologic conditions, including pulmonary edema and vessel inflammation. In endothelial monolayers, TGF-beta1 increases permeability, and a number of studies have demonstrated the alteration of cell-cell contacts by TGF-beta1. We hypothesized that focal adhesion complexes also likely contribute to alterations in endothelial permeability. We examined early signal transduction events associated with rapid changes in monolayer permeability and the focal adhesion complex of bovine pulmonary artery endothelial cells. Western blotting revealed rapid tyrosine phosphorylation of focal adhesion kinase (FAK) and Src kinase in response to TGF-beta1; inhibition of both of these kinases using pp2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), ameliorates TGF-beta1-induced monolayer permeability. Activation of FAK/Src requires activation of the epidermal growth factor receptor downstream of the TGF-beta receptors, and is blocked by the epidermal growth factor receptor inhibitor AG1478. Immunohistochemistry showed that actin and the focal adhesion proteins paxillin, vinculin, and hydrogen peroxide-inducible clone-5 (Hic-5) are rearranged in response to TGF-beta1; these proteins are released from focal adhesion complexes. Rearrangement of paxillin and vinculin by TGF-beta1 is not blocked by the FAK/Src inhibitor, pp2, or by SB431542 inhibition of the TGF-beta type I receptor, anaplastic lymphoma kinase 5; however, pp1 (4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which inhibits both type I and type II TGF-beta receptors, does block paxillin and vinculin rearrangement. Hic-5 protein rearrangement requires FAK/Src activity. Together, these results suggest that TGF-beta1-induced monolayer permeability involves focal adhesion and cytoskeletal rearrangement through both FAK/Src-dependent and -independent pathways.

The focal adhesion protein Hic-5 is highly expressed in the rat myometrium during late pregnancy and labour and co-localizes with FAK

BackgroundMyometrial growth and remodeling of the cytoskeleton and focal adhesions during late pregnancy may be critical aspects of myometrial activation and thus labour. Yet our understanding of these aspects is inhibited by the paucity of information concerning the components of focal adhesions in the myometrium. The focal adhesion protein hydrogen peroxide-inducible clone-5 (Hic-5) has recently been found in mononuclear smooth muscle but was not examined in the myometrium during pregnancy. Thus, the goal of this study was to characterize Hic-5 mRNA and protein expression in the rat myometrium during pregnancy and labour.MethodsRat myometrium samples were obtained from non-pregnant animals, pregnant animals on days (d) 6, 12, 15, 17, 19, 21, 22, 23 (active labour) and 1 day postpartum (PP). In addition, myometrium samples were collected from rats within a progesterone-delayed labour paradigm. Hic-5 mRNA expression was analyzed by Northern blot analysis while Hic-5 protein expression was examined by immunoblot and immunofluorescence analysis.ResultsHic-5 mRNA expression on d15, d19 and d21 was found to be significantly elevated compared to d6 and d12 of pregnancy and expression on d23 was significantly elevated over d6 (p < 0.05). Immunofluorescence analysis demonstrated that detection of Hic-5 protein in the circular muscle layer appeared to increase from d17 onwards, except PP, and Hic-5 was detectable in the cell cytoplasm and more continuously associated with myometrial cell membranes. In the longitudinal muscle layer Hic-5 was readily detectable by d15 and thereafter and primarily associated at myometrial cell membranes. Co-immunofluorescence analysis of potential Hic-5 and focal adhesion kinase (FAK) association in situ demonstrated a limited level of co-localization on d19, d23 and PP in the circular muscle layer while in the longitudinal muscle layer Hic-5 and FAK were readily co-localized at myometrial cell membranes.ConclusionHic-5 is highly expressed in the rat myometrium during late pregnancy and labour and co-localizes with FAK in situ. Our results are consistent with a potential role for Hic-5 in focal adhesion remodeling in the rat myometrium during late pregnancy.

Hic-5 promotes endothelial cell migration to lysophosphatidic acid

Endothelial cell migration is critical for proper blood vessel development. Signals from growth factors and matrix proteins are integrated through focal adhesion proteins to alter cell migration. Hydrogen peroxide-inducible clone 5 (Hic-5), a paxillin family member, is enriched in the focal adhesions in bovine pulmonary artery endothelial (BPAE) cells, which migrate to lysophosphatidic acid (LPA) on denatured collagen. In this study, we investigate the role of Hic-5 in LPA-stimulated endothelial cell migration. LPA recruits Hic-5 to the focal adhesions and to the pseudopodia in BPAE cells plated on collagen, suggesting that recruitment of Hic-5 to focal adhesions is associated with endothelial cell migration. Knockdown of endogenous Hic-5 significantly decreases migration toward LPA, confirming involvement of Hic-5 in migration. To address the role of Hic-5 in endothelial cell migration, we exogenously expressed wild-type (WT) Hic-5 and green fluorescent protein Hic-5 C369A/C372A (LIM3 mutant) constructs in BPAE cells. WT Hic-5 expression increases chemotaxis of BPAE cells to LPA, whereas migration toward LPA of the green fluorescent protein Hic-5 C369A/C372A-expressing cells is similar to that shown in vector control cells. Additionally, ERK phosphorylation is enhanced in the presence of LPA in WT Hic-5 cells. A pharmacological inhibitor of MEK activity inhibits LPA-stimulated WT Hic-5 cell migration and ERK phosphorylation, suggesting Hic-5 enhances migration via MEK activation of ERK. Together, these studies indicate that Hic-5, a focal adhesion protein in endothelial cells, is recruited to the pseudopodia in the presence of LPA and enhances migration.

The Multiple LIM Domain-Containing Adaptor Protein Hic-5 Synaptically Colocalizes and Interacts with the Dopamine Transporter

The Na+/Cl--dependent dopamine transporter (DAT) is critical in terminating dopaminergic transmission by removing the transmitter away from the synapse. Several lines of evidence suggest that transporter-interacting proteins may play a role in DAT function and regulation. In this report, using the yeast two-hybrid system, we have identified a novel interaction between DAT and the multiple Lin-11, Isl-1, and Mec-3 (LIM) domain-containing adaptor protein Hic-5. This association involves the N-terminal portion of the intracellular tail of DAT and the LIM region of Hic-5. In human embryonic kidney 293 cells, Hic-5 colocalizes with DAT at polarized sites and reduces DAT uptake activity through a mechanism involving a decrease in the cell-surface levels of the transporter. A fragment of Hic-5 containing the LIM domains is sufficient to bind DAT but lacks the ability to inhibit transporter activity. In addition, the LIM fragment prevents the effect of the full-length Hic-5 on DAT localization and function. In the brain, Hic-5 protein is expressed in the cerebral cortex, hippocampus, hypothalamus, cerebellum, and striatum, suggesting a role for this protein in the nervous system. The association of the endogenous Hic-5 and DAT proteins was confirmed biochemically by coimmunoprecipitation from brain striatal extracts. Moreover, immunostaining of rat midbrain neurons in culture revealed a presynaptic colocalization of Hic-5 and DAT. Because Hic-5 has been shown to interact with several signaling molecules, including the nonreceptor protein tyrosine kinases focal adhesion kinase and Fyn, this raises the possibility that this adaptor protein may link DAT to intracellular signaling pathways.

The multiple LIM domain-containing adaptor protein Hic-5 synaptically colocalizes and interacts with the dopamine transporter.

The Na+/Cl--dependent dopamine transporter (DAT) is critical in terminating dopaminergic transmission by removing the transmitter away from the synapse. Several lines of evidence suggest that transporter-interacting proteins may play a role in DAT function and regulation. In this report, using the yeast two-hybrid system, we have identified a novel interaction between DAT and the multiple Lin-11, Isl-1, and Mec-3 (LIM) domain-containing adaptor protein Hic-5. This association involves the N-terminal portion of the intracellular tail of DAT and the LIM region of Hic-5. In human embryonic kidney 293 cells, Hic-5 colocalizes with DAT at polarized sites and reduces DAT uptake activity through a mechanism involving a decrease in the cell-surface levels of the transporter. A fragment of Hic-5 containing the LIM domains is sufficient to bind DAT but lacks the ability to inhibit transporter activity. In addition, the LIM fragment prevents the effect of the full-length Hic-5 on DAT localization and function. In the brain, Hic-5 protein is expressed in the cerebral cortex, hippocampus, hypothalamus, cerebellum, and striatum, suggesting a role for this protein in the nervous system. The association of the endogenous Hic-5 and DAT proteins was confirmed biochemically by coimmunoprecipitation from brain striatal extracts. Moreover, immunostaining of rat midbrain neurons in culture revealed a presynaptic colocalization of Hic-5 and DAT. Because Hic-5 has been shown to interact with several signaling molecules, including the nonreceptor protein tyrosine kinases focal adhesion kinase and Fyn, this raises the possibility that this adaptor protein may link DAT to intracellular signaling pathways.

Accumulation of Focal Adhesion Protein Hic-5 in the Nucleus by Hydrogen Peroxide.

Reactive oxygen species (ROS) are produced in cultured cells upon stimulation with cytokines and tumor promoters, and participate in signal transduction pathways leading to gene expression. The hic-5 gene was isolated as a hydrogen peroxide-inducible clone by differential screening, and was shown to encode a paxillin-related protein localized in the focal adhesion plaques. In mouse osteoblastic and fibroblastic cells treated with low concentrations of hydrogen peroxide, Hic-5 protein was found to be reversibly localized to the nucleus within 30 min. Hic-5 was also translocated to the nucleus in cells treated with leptomycin B, an inhibitor of nuclear export of proteins. Hic-5 protein with mutations in the nuclear export signal and the LIM-3 domain accumulated in the nucleus. These results indicated that Hic-5 shuttles between the cytoplasm and the nucleus, and may participate both in surface signals and in gene expression.

Specific decrease in the level of Hic-5, a focal adhesion protein, during immortalization of mouse embryonic fibroblasts, and its association with focal adhesion kinase.

Hic-5 is a paxillin homologue with four LIM domains in its C-terminal region, localized mainly in focal adhesions in normal fibroblasts. Hic-5 is also known to associate with focal adhesion kinase (FAK) or the related CAKbeta, and with vinculin. In the present study, we examined changes in Hic-5 and paxillin protein levels in primary mouse embryo fibroblasts (MEF) during mortal and immortal stages. The Hic-5 level was markedly decreased when cells became immortalized, whereas that of paxillin was increased. The vinculin level was not changed significantly. Hic-5 was mainly localized in focal adhesion plaques of mortal MEF but was localized in the nuclear periphery in the immortalized MEF; the number of focal adhesion plaques was decreased in these cells. Mouse Hic-5 contains three LD domains in its N-terminal half, and the first LD domain (LD1) appears to be involved in interaction with FAK. However, this interaction was not essential for recruitment of Hic-5 to focal adhesions, since its subcellular localization was similar in FAK(-/-) cells. Forced expression of Hic-5 decreased colony forming ability of MEF from FAK(+/+) mice, but not of FAK(-/-) cells. These observations suggested the involvement of Hic-5 in determination of cellular proliferative capacity in collaboration with other cytoskeletal components.

Hic-5, a Paxillin Homologue, Binds to the Protein-tyrosine Phosphatase PEST (PTP-PEST) through Its LIM 3 Domain

The Hic-5 protein is encoded by a transforming growth factor-beta1- and hydrogen peroxide-inducible gene, hic-5, and has striking similarity to paxillin, especially in their C-terminal LIM domains. Like paxillin, Hic-5 is localized in focal adhesion plaques in association with focal adhesion kinase in cultured fibroblasts. We carried out yeast two-hybrid screening to identify cellular factors that form a complex with Hic-5 using its LIM domains as a bait, and we identified a cytoplasmic tyrosine phosphatase (PTP-PEST) as one of the partners of Hic-5. These two proteins are associated in mammalian cells. From in vitro binding experiments using deletion and point mutations, it was demonstrated that the essential domain in Hic-5 for the binding was LIM 3. As for PTP-PEST, one of the five proline-rich sequences found on PTP-PEST, Pro-2, was identified as the binding site for Hic-5 in in vitro binding assays. Paxillin also binds to the Pro-2 domain of PTP-PEST. In conclusion, Hic-5 may participate in the regulation of signaling cascade through its interaction with distinct tyrosine kinases and phosphatases.

The LIM domains of hic-5 protein recognize specific DNA fragments in a zinc-dependent manner in vitro.

hic-5 protein is a member of the LIM protein family, containing four LIM domains in its C-terminal region. It is mainly localized in focal adhesions and shows striking similarity to paxillin in its LIM domains, although the function of these LIM domains has remained elusive. In the present study, we found that full-length and the C-terminal half of hic-5 protein, including four LIM domains, bound to DNA in a zinc-dependent manner in vitro . Mouse genomic fragments that specifically bound to the hic-5 protein were isolated by successive rounds of hic-5 protein-DNA complex immunoprecipitation and PCR amplification. Seven independent clones were isolated, which contained high amounts of G+A and/or a long A/T tract. A DNA binding protein blot assay revealed the specificity of the interaction between hic-5 protein and the DNA fragment. Using a series of truncated forms of the hic-5 LIM domains, each of the four LIM domains was found to contribute to DNA binding in a distinctive manner.

Characterization of the TGF beta 1-inducible hic-5 gene that encodes a putative novel zinc finger protein and its possible involvement in cellular senescence.

Transforming growth factor (TGF) beta 1 is a potent cytokine that inhibits the growth of several types of cells. Our earlier study suggested that the mouse osteoblastic cell line, MC3T3-E1, was sensitive to growth inhibition by TGF beta 1 and that this effect was partly mediated by H2O2. To identify the molecules that participate in the negative regulation of growth by these stimuli, we carried out differential screening of cDNA libraries and isolated a set of genes induced by TGF beta 1. Among the clones isolated, one originally named tsc-5 was found to be induced by H2O2 as well as TGF beta 1. Analysis of this cDNA renamed hic (hydrogen peroxide-inducible clone)-5 suggested that Hic-5 protein has four LIM motifs, each of which contained two (or one) putative zinc fingers. The expression of hic-5 mRNA was repressed in Ki-ras-transformed mouse fibroblasts and in several cell lines established from human tumor. On the other hand, its expression was augmented in the in vitro senescent process of human diploid fibroblasts. Among the mouse organs examined, hic-5 was highly expressed in the lung and spleen. Finally, a colony formation assay using an hic-5 expression vector driven by the cytomegalovirus promoter suggested that hic-5 overexpression had a cytostatic effect on cellular growth, depending upon the cell type. Although the relationship between hic-5 function and the signal transduction pathway of TGF beta 1 remains unresolved, these results implied that hic-5 has some role in the growth-inhibitory pathway associated with in vitro senescence, and that down-regulation of hic-5 contributes to tumorigenesis.