Hiroyasu Inada

Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD

Keratin 8 and 18 (K8/18) are the major components of intermediate filament (IF) proteins of simple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tumor necrosis factor (TNF)–induced cell death. We have now identified human TNF receptor type 1 (TNFR1)–associated death domain protein (TRADD) to be the K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound K18 and K14, type I (acidic) keratins. The COOH-terminal region of TRADD interacted with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH2 terminus (amino acids 1–270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation. These results indicate that K18 may sequester TRADD to attenuate interactions between TRADD and activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.

Tenascin-C Regulates Recruitment of Myofibroblasts during Tissue Repair after Myocardial Injury

Tenascin-C (TN-C) is an extracellular matrix molecule that is expressed during wound healing in various tissues. Although not detectable in the normal adult heart, it is expressed under pathological conditions. Previously, using a rat model, we found that TN-C was expressed during the acute stage after myocardial infarction and that alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts appeared in TN-C-positive areas. In the present study, we examined whether TN-C controls the dynamics of myofibroblast recruitment and wound healing after electrical injury to the myocardium of TN-C knockout (TNKO) mice compared with wild-type (WT) mice. In TNKO mice, myocardial repair seemed to proceed normally, but the appearance of myofibroblasts was delayed. With cultured cardiac fibroblasts, TN-C significantly accelerated cell migration, alpha-SMA expression, and collagen gel contraction but did not affect proliferation. Using recombinant fragments of murine TN-C, the functional domain responsible for promoting migration of cardiac fibroblasts was mapped to the conserved fibronectin type III (FNIII)-like repeats and the fibrinogen (Fbg)-like domain. Furthermore, alternatively spliced FNIII and Fbg-like domains proved responsible for the up-regulation of alpha-SMA expression. These results indicate that TN-C promotes recruitment of myofibroblasts in the early stages of myocardial repair by stimulating cell migration and differentiation.

Heme oxygenase-1 gene induction as an intrinsic regulation against delayed cerebral vasospasm in rats

Delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) causes cerebral ischemia and infarction. To date, the pathogenesis and gene expression associated with vasospasm remain poorly understood. The present study used fluorescent differential display to identify differentially expressed genes in a rat model of SAH. By using quantitative RT-PCR, we found that heme oxygenase-1 (HO-1) mRNA was prominently induced in the basilar artery and modestly in brain tissue in a rat vasospasm model. A significant correlation was observed between the degree of vasospasm and HO-1 mRNA levels in the basilar arteries exhibiting vasospasm. Intracisternal injection of antisense HO-1 oligodeoxynucleotide (ODN) significantly delayed the clearance of oxyhemoglobin and deoxyhemoglobin from the subarachnoid space and aggravated angiographic vasospasm. Antisense HO-1 ODN inhibited HO-1 induction in the basilar arteries but not in the whole brain tissue. This phenomenon was not observed in the nontreated, sense HO-1 ODN-treated, or scrambled ODN-treated arteries. We report the protective effects of HO-1 gene induction in cerebral vasospasm after SAH, a finding that should provide a novel therapeutic approach for cerebral vasospasm.

Involvement of Large Tenascin-C Splice Variants in Breast Cancer Progression

Alternative splicing of fibronectin-like type III (FNIII) repeats of tenascin-C (Tn-C) generates a number of splice variants. The distribution of large variants, typical components of provisional extracellular matrices that are up-regulated during tumor stroma remodeling, was here studied by immunoblotting and immunohistochemistry using a monoclonal antibody against the FNIII B domain (named 4C8MS) in a series of human breast cancers. Large Tn-C variants were found at only low levels in normal breast tissues, but were highly expressed at invading sites of intraductal cancers and in the stroma of invasive ductal cancers, especially at invasion fronts. There was a positive correlation between the expression of large Tn-C variants and the cell proliferation rate determined by immunolabeling of the Ki-67 antigen. Of the Tn-C recombinant fragments (all FNIII repeats or mFNIII FL, the conserved FNIII domain only, the epidermal growth factor-like domain, and the fibrinogen-like domain) which were expressed by CHO-K1 cells transfected with mouse Tn-C cDNAs, only the mFNIII FL enhanced in vitro migration and mitotic activity of mammary cancer cells derived from a Tn-C-null mouse. Addition of 4C8MS blocked the function of mFNIII FL. These findings provide strong evidence that the FNIII alternatively spliced region has important roles in tumor progression of breast cancer.

Tenascin-C upregulates matrix metalloproteinase-9 in breast cancer cells: Direct and synergistic effects with transforming growth factor β1

Tenascin‐C (TN‐C) and matrix metalloproteinases (MMPs) are highly expressed in cancer tissues and probably promote cell migration during cancer progression. TN‐C and MMPs are often co‐localized in areas of active tissue remodeling in pathologic conditions, suggesting reciprocal regulation. To investigate whether TN‐C regulates MMPs expression in cancer cells, we first exposed mammary cancer cells derived from TN‐C‐deficient mice to TN‐C and examined MMPs expression. TN‐C was then compared with fibronectin (FN), laminin (LN), basic fibroblast growth factor (b‐FGF) and transforming growth factor‐beta1 (TGF‐β1). Results of endpoint RT‐PCR, quantitative real‐time RT‐PCR and gelatin zymography demonstrated that TN‐C, strongly and dose dependently, upregulates MMP‐9 expression in murine mammary cancer cells. TN‐C weakly induced MMP‐2, MMP‐3 and MMP‐13. FN and LN induced MMP‐9 to lesser extents compared with TN‐C. b‐FGF had no effect on MMP‐9 expression. TGF‐β1 induced MMP‐9 expression in a dose‐dependent manner, and this induction was significantly enhanced by addition of TN‐C. TN‐C and TGF‐β1 also upregulated MMP‐9 expression in the human breast cancer cell line MDA‐MB‐231. Neutralization with specific anti‐TGF‐β1 antibody showed decreased expression of MMP‐9, indicating that TGF‐β controls the baseline MMP‐9 expression by a direct autocrine mechanism. Under neutralization of TGF‐β, addition of TN‐C still upregulated MMP‐9. Conversely, neutralization of endogenous TN‐C (in a TN‐C‐positive mammary cancer cell line) downregulated TGF‐β‐induced MMP‐9 expression. Thus, TN‐C induces MMP‐9 expression directly and by collaboration with TGF‐β. These findings reveal a novel role of TN‐C in breast cancer progression.

Identification of Mrj, a DnaJ/Hsp40 Family Protein, as a Keratin 8/18 Filament Regulatory Protein

To elucidate the function of keratins 8 and 18 (K8/18), major components of the intermediate filaments of simple epithelia, we searched for K8/18-binding proteins by screening a yeast two-hybrid library. We report here that human Mrj, a DnaJ/Hsp40 family protein, directly binds to K18. Among the interactions between DnaJ/Hsp40 family proteins and various intermediate filament proteins that we tested using two-hybrid methods, Mrj specifically interacted with K18. Immunostaining with anti-Mrj antibody showed that Mrj colocalized with K8/18 filaments in HeLa cells. Mrj was immunoprecipitated not only with K18, but also with the stress-induced and constitutively expressed heat shock protein Hsp/c70. Mrj bound to K18 through its C terminus and interacted with Hsp/c70 via its N terminus, which contains the J domain. Microinjection of anti-Mrj antibody resulted in the disorganization of K8/18 filaments, without effects on the organization of actin filaments and microtubules. Taken together, these results suggest that Mrj may play an important role in the regulation of K8/18 filament organization as a K18-specific co-chaperone working together with Hsp/c70.

Deficiency of tenascin-C attenuates liver fibrosis in immune-mediated chronic hepatitis in mice

Tenascin‐C (TNC), an extracellular matrix glycoprotein, is upregulated in chronic liver disease. Here, we investigated the contribution of TNC to liver fibrogenesis by comparing immune‐mediated hepatitis in wild‐type (WT) and TNC‐null (TNKO) mice. Eight‐week‐old BALB/c mice received weekly intravenous injections of concanavalin A to induce hepatitis, and were sacrificed one week after the 3rd, 6th, 9th, and 12th injections. In WT livers, immunohistochemical staining revealed a gradual increase in TNC deposition. TNC mRNA levels also increased sequentially and peaked after the 9th injection. Collagen deposition, stained with picrosirius red, was significantly less intense in TNKO mice than in WT mice, and procollagen I and III transcripts were significantly upregulated in WT mice compared with TNKO mice. Inflammatory infiltrates were most prominent after the 3rd‐6th injections in both groups and were less intense in TNKO mice than in WT mice. Interferon‐γ, tumour necrosis factor‐α, and interleukin‐4 mRNA levels were significantly higher in WT mice than in TNKO mice, while activated hepatic stellate cells (HSCs) and myofibroblasts, a cellular source of TNC and procollagens, were more common in WT livers. Transforming growth factor (TGF)‐β1 mRNA expression was significantly upregulated in WT mice, but not in TNKO mice. In conclusion, TNC can promote liver fibrogenesis through enhancement of inflammatory response with cytokine upregulation, HSC recruitment, and TGF‐β expression during progression of hepatitis to fibrosis. Copyright

Eplerenone attenuates myocardial fibrosis in the angiotensin II-induced hypertensive mouse: involvement of tenascin-C induced by aldosterone-mediated inflammation.

Tenascin-C is an extracellular matrix glycoprotein that is supposed to be a profibrotic molecule in various fibrogenic processes. To elucidate its significance for myocardial fibrosis in the hypertensive heart, we used a mouse model with infusion of angiotensin II and examined results by histology, immunohistochemistry, in situ hybridization, and quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Angiotensin II treatment elevated blood pressure and expression of tenascin-C by interstitial fibroblasts in perivascular fibrotic lesions, and angiotensin II infusion caused accumulation of macrophages. It also upregulated expression of collagen Iα2; IIIα1; and proinflammatory/profibrotic mediators including transforming growth factor beta (TGFβ), platelet-derived growth factor alpha (PDGF-A), PDGF-B, and PDGF-receptor α, but not IL-1β and PDGF-receptor β, in the myocardium. Treatment with an aldosterone receptor antagonist, eplerenone, significantly attenuated angiotensin II-induced fibrosis, expression of tenascin-C, and inflammatory changes without affecting the blood pressure level. In vitro, neither eplerenone nor aldosterone exerted any influence on tenascin-C expression of cardiac fibroblasts, whereas angiotensin II, TGF-β1, and PDGF significantly upregulated expression of tenascin-C. These results suggest that, in the angiotensin II-induced hypertensive mouse heart: (1) tenascin-C may be involved in the progression of cardiac fibrosis and (2) aldosterone may elicit inflammatory reactions in myocardium, which might, in turn, induce tenascin-C synthesis of fibroblasts through at least 2 pathways mediated by TGF-β and PDGF-A-B/PDGF-receptor α.

Densin‐180, a synaptic protein, links to PSD‐95 through its direct interaction with MAGUIN‐1

Background: Densin‐180, a brain‐specific protein highly concentrated at the postsynaptic density (PSD), belongs to the LAP [leucine‐rich repeats and PSD‐95/Dlg‐A/ZO‐1 (PDZ) domains] family of proteins, some of which play fundamental roles in the establishment of cell polarity.

Co-stimulation of human breast cancer cells with transforming growth factor-β and tenascin-C enhances matrix metalloproteinase-9 expression and cancer cell invasion

Transforming growth factor‐β (TGF‐β), tenascin‐C (TN‐C) and matrix metalloproteinases (MMPs) have been demonstrated independently to be associated with disease progression and poor prognosis in patients with breast cancer. The present study explored effects of TGF‐β and TN‐C on MMP‐9 expression and cancer invasion. An experimental study was designed to analyse MDA‐MB‐231 breast cancer cells, known for their high invasiveness, after stimulation with TGF‐β1 and/or TN‐C. TGF‐β1 stimulated TN‐C expression in the cells. Co‐stimulation of MDA‐MB‐231 cells with TN‐C and TGF‐β increased MMP‐9 expression at both the gene (28‐fold) and the protein levels. The in vitro invasion also increased (4‐fold). GM6001 inhibited the invasion induced by the co‐stimulation. The combined effect of TN‐C and TGF‐β resulted in enhanced MMP‐9 expression and cancer invasion in vitro.