scholarly journals JNK and PI3K differentially regulate MMP-2 and MT1-MMP mRNA and protein in response to actin cytoskeleton reorganization in endothelial cells

2006 ◽  
Vol 291 (4) ◽  
pp. C579-C588 ◽  
Author(s):  
Eric Ispanovic ◽  
Tara L. Haas
Keyword(s):  
Endothelial Cells ◽  
Matrix Metalloproteinase ◽  
Actin Cytoskeleton ◽  
Mrna Expression ◽  
Protein Production ◽  
Critical Role ◽  
Cytochalasin D ◽  
Matrix Metalloproteinase 2 ◽  
Downstream Target ◽  
Protein Levels

Increased production and activation of matrix metalloproteinase-2 (MMP-2) are critical events in skeletal muscle angiogenesis and are known to occur in response to mechanical stresses. We hypothesized that reorganization of the actin cytoskeleton would increase endothelial cell production and activation of MMP-2 and that this increase would require a MAPK-dependent signaling pathway in endothelial cells. The pharmacological actin depolymerization agent cytochalasin D increased expression of MMP-2 and membrane type 1-matrix metalloproteinase (MT1-MMP) mRNA, and this was reduced significantly in the presence of the JNK inhibitor SP600125. Activation of JNK by anisomycin was sufficient to induce expression of both MMP-2 and MT1-MMP mRNA in quiescent cells. Downregulation of c-Jun, a downstream target of JNK, with small interference (si)RNA inhibited MMP-2 expression in response to anisomycin. Inhibition of phosphoinositide 3-kinase (PI3K), but not JNK, significantly decreased the amount of active MMP-2 following cytochalasin D stimulation with a concurrent decrease in MT1-MMP protein. Physiological reorganization of actin occurs during VEGF stimulation. VEGF-induced MMP-2 protein production and activation, as well as MT1-MMP protein production, depended on PI3K activity. VEGF-induced MMP-2 mRNA expression was reduced by inhibition of JNK or by treatment with c-Jun siRNA. In summary, our results provide novel insight into the signaling cascades initiated in the early stages of angiogenesis through the reorganization of the actin cytoskeleton and demonstrate a critical role for JNK in regulating MMP-2 and MT1-MMP mRNA expression, whereas PI3K regulates protein levels of both MMP-2 and MT1-MMP.

Fli1 Deficiency Induces CXCL6 Expression in Dermal Fibroblasts and Endothelial Cells, Contributing to the Development of Fibrosis and Vasculopathy in Systemic Sclerosis

2017 ◽  
Vol 44 (8) ◽  
pp. 1198-1205 ◽  
Author(s):  
Takashi Taniguchi ◽  
Yoshihide Asano ◽  
Kouki Nakamura ◽  
Takashi Yamashita ◽  
Ryosuke Saigusa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Systemic Sclerosis ◽  
Mrna Expression ◽  
Target Genes ◽  
Critical Role ◽  
Dermal Fibroblasts ◽  
Vascular Involvement ◽  
Digital Ulcers ◽  
Mrna Levels ◽  
Predisposing Factor

Objective.CXCL6, a chemokine with proangiogenic property, is reported to be involved in vasculopathy associated with systemic sclerosis (SSc). We investigated the contribution of CXCL6 to SSc development by focusing on the association of friend leukemia virus integration 1 (Fli1) deficiency, a potential predisposing factor of SSc, with CXCL6 expression and clinical correlation of serum CXCL6 levels.Methods.mRNA levels of target genes and the binding of Fli1 to the CXCL6 promoter were evaluated by quantitative reverse transcription-PCR and chromatin immunoprecipitation, respectively. Serum CXCL6 levels were determined by ELISA.Results.FLI1 siRNA significantly enhanced CXCL6 mRNA expression in human dermal fibroblasts and human dermal microvascular endothelial cells, while Fli1 haploinsufficiency significantly suppressed CXCL6 mRNA expression in murine peritoneal macrophages stimulated with lipopolysaccharide. Supporting a critical role of Fli1 deficiency to induce SSc-like phenotypes, CXCL6 mRNA expression was higher in SSc dermal fibroblasts than in normal dermal fibroblasts. Importantly, Fli1 bound to the CXCL6 promoter in dermal fibroblasts, endothelial cells, and THP-1 cells. In patients with SSc, serum CXCL6 levels correlated positively with the severity of dermal and pulmonary fibrosis and were elevated in association with cardiac and pulmonary vascular involvement and cutaneous vascular symptoms, including Raynaud phenomenon, digital ulcers (DU)/pitting scars, and telangiectasia. Especially, serum CXCL6 levels were associated with DU/pitting scars and heart involvement by multiple regression analysis.Conclusion.CXCL6 expression is upregulated by Fli1 deficiency in fibroblasts and endothelial cells, potentially contributing to the development of fibrosis and vasculopathy in the skin, lung, and heart of SSc.

scholarly journals Association of Matrix Metalloproteinase-2 mRNA Expression with Subtypes of Pediatric Cholesteatoma

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Taichi Kan ◽  
Hiromi Ueda ◽  
Taishi Takahara ◽  
Yoshimasa Tsuchiya ◽  
Mayuko Kishimoto ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Matrix Metalloproteinase ◽  
Mrna Expression ◽  
Bone Destruction ◽  
Expression Level ◽  
Matrix Metalloproteinase 2 ◽  
Mrna Expression Level ◽  
Congenital Cholesteatoma ◽  
Rna In Situ Hybridization

Objective. Cholesteatoma is a clinically heterogeneous disease, with some patients showing spontaneous regression, while others experiencing an aggressive, lethal disease. Cholesteatoma in children can be divided into two types: congenital and acquired. Identifying good prognostic markers is needed to help select patients who will require immediate surgical intervention. Matrix metalloproteinase-2 (MMP2) was previously reported to play an important role in cholesteatoma progression, by promoting bone destruction and keratinocyte infiltration. Herein, we analyzed MMP2 mRNA expression level in cholesteatoma using RNA-in situ hybridization in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Methods. Sixty patients with cholesteatoma under 15 years old, who underwent their primary surgery at Aichi Medical University’s Otolaryngology Department, were analyzed for MMP2 expression level, using RNA-in situ hybridization. Results. There were no significant differences in MMP2 mRNA expression level between congenital cholesteatoma and acquired cholesteatomas. In congenital cholesteatoma, higher MMP2 signals were observed in the open type than in the closed type ( p < 0.001 ). In acquired cholesteatoma, higher MMP2 signals were observed in the pars tensa than in the pars flaccida ( p < 0.001 ). MMP2 mRNA expression level was almost exclusively found in the fibroblasts or in the inflammatory cells in the stroma, but not in the epithelium. Conclusion. Our study reveals that MMP2 mRNA expression level is strongly associated with the subtypes of cholesteatoma. The findings suggest that the level of expression of MMP2 mRNA may be related to the pathogenesis and aggressive features of cholesteatoma.

Activation of pro-(matrix metalloproteinase-2) (pro-MMP-2) by thrombin is membrane-type-MMP-dependent in human umbilical vein endothelial cells and generates a distinct 63 kDa active species

2001 ◽  
Vol 357 (1) ◽  
pp. 107-115 ◽  
Author(s):  
Marc A. LAFLEUR ◽  
Morley D. HOLLENBERG ◽  
Susan J. ATKINSON ◽  
Vera KNÄUPER ◽  
Gillian MURPHY ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Matrix Metalloproteinase ◽  
Umbilical Vein ◽  
Coagulation Cascade ◽  
Matrix Metalloproteinase 2 ◽  
Intermediate Form ◽  
Dependent Manner ◽  
Human Umbilical Vein ◽  
Membrane Type ◽  
Umbilical Vein Endothelial Cells

Thrombin, a critical enzyme in the coagulation cascade, has also been associated with angiogenesis and activation of the zymogen form of matrix metalloproteinase-2 (MMP-2 or gelatinase-A). We show that thrombin activated pro-MMP-2 in a dose- and time-dependent manner in cultured human umbilical-vein endothelial cells (HUVECs) to generate a catalytically active 63kDa protein that accumulated as the predominant form in the conditioned medium. This 63kDa thrombin-activated MMP-2 is distinct from the 62kDa species found following concanavalin A or PMA stimulated pro-MMP-2 activation. Hirudin and leupeptin blocked thrombin-induced pro-MMP-2 activation, demonstrating that the proteolytic activity of thrombin is essential. However, activation was also dependent upon membrane-type-MMP (MT-MMP) action, since it was blocked by EDTA, o-phenanthroline, hydroxamate metalloproteinase inhibitors, tissue inhibitor of metalloproteinase-2 (TIMP-2) and TIMP-4, but not TIMP-1. Thrombin inefficiently cleaved recombinant 72kDa pro-MMP-2, but efficiently cleaved the 64kDa MT-MMP-processed intermediate form in the presence of cells. Thrombin also rapidly (within 1h) increased cellular MT-MMP activity, and at longer time points (>6h) it increased expression of MT1-MMP mRNA and protein. Thus signalling via proteinase-activated receptors (PARs) may play a role in thrombin-induced MMP-2 activation, though this does not appear to involve PAR1, PAR2, or PAR4 in HUVECs. These results indicate that in HUVECs the activation of pro-MMP-2 by thrombin involves increased MT-MMP activity and preferential cleavage of the MT-MMP-processed 64kDa MMP-2 form in the presence of cells. The integration of these proteinase systems in the vascular endothelium may be important during thrombogenesis and tissue remodelling associated with neovascularization.

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