Biomedical subjects

Alex Y Strongin

Publications and source records attributed to Alex Y Strongin.

43 records · Page 3Linked to original sources

PubMed2002

Unconventional activation mechanisms of MMP-26, a human matrix metalloproteinase with a unique PHCGXXD cysteine-switch motif.

ProMMP-26 has the unique Pro-His(81)-Cys-Gly-Xaa-Xaa-Asp cysteine-switch motif that discriminates this protease from all other matrix metalloproteinases (MMPs) known so far. The conserved, free cysteine residue of the conventional PRCXXPD sequence interacts with the zinc ion of the catalytic domain and provides the fourth coordination site for the catalytic zinc, thereby preventing latent proMMPs from becoming active. MMPs become functionally active when proteolytic cleavage releases the prodomain and the PRCXXPD sequence and exposes the zinc atom. Here, we report that the Pro-His(81)-Cys-Gly-Xaa-Xaa-Asp motif is not functional in proMMP-26 and consequently is not involved in the activation mechanisms. Organomercurial treatment failed to activate proMMP-26. The autolytic Lys-Lys-Gln(59) downward arrow Gln(60)-Phe-His cleavage upstream of the Pro-His(81)-Cys-Gly-Xaa-Xaa-Asp motif induced the proteolytic activity of recombinant proMMP-26 whereas any further cleavage inactivated the enzyme. The His(81) --> Arg(81) mutation restored the conventional cysteine-switch sequence in the prodomain but failed to induce the cysteine-switch activation mechanism. These data and computer modeling studies allowed us to hypothesize that the presence of His(81) significantly modified the fold of proMMP-26, abolished the functionality of the cysteine-switch motif, and stimulated an alternative intramolecular activation pathway of the proenzyme.

Amino Acid Motifs↗

PubMed2002

Up-regulation of vascular endothelial growth factor by membrane-type 1 matrix metalloproteinase stimulates human glioma xenograft growth and angiogenesis.

Membrane-type (MT) 1 matrix metalloproteinase (MMP) is up-regulated in many tumor types and has been implicated in tumor progression and metastasis. MT1-MMP is critical for pericellular degradation of the extracellular matrix, thereby promoting tumor cell invasion and dissemination. To grow efficiently in vivo, tumor cells induce angiogenesis in both primary solid tumors and metastatic foci. The present study describes a functional link between the expression of MT1-MMP and vascular endothelial growth factor (VEGF) production in human glioma U251 xenografts in athymic mice. To investigate the effects of MT1-MMP on VEGF expression, U251 cells were stably transfected with MT1-MMP to generate the U-MT cell line overexpressing the enzyme. In vitro, the U-MT cells had an increased rate of proliferation and migration as well as the ability to activate the MMP-2 proenzyme and directionally remodel a three-dimensional collagen matrix. These findings suggested higher tumorigenicity of U-MT cells relative to the vector-control U-neo cells. In agreement with the in vitro data, U-MT xenografts in BALB/c nu/nu mice displayed markedly increased growth rates and elevated levels of angiogenesis. In contrast, U-neo cells formed small, minimally vascularized tumors. The elevated angiogenesis in U-MT xenografts was associated with an up-regulation of VEGF expression in tumor cells. In addition, U-MT cells in vitro secreted twice as much VEGF as the control cells. GM6001, a hydroxamate inhibitor of MMP activity, down-regulated the production of VEGF in U-MT cells to the levels observed in the U-neo control. Our results demonstrate that the enhanced tumorigenicity of glioma cells overexpressing MT1-MMP involves stimulation of angiogenesis through the up-regulation of VEGF production.

Animals↗

PubMed2002

Gelatin zymography and substrate cleavage assays of matrix metalloproteinase-2 in breast carcinoma cells overexpressing membrane type-1 matrix metalloproteinase.

Gelatin zymography is the common method for examining matrix metalloproteinase-2 (MMP-2) in cells and media samples. Activation of the latent MMP-2 zymogen involves its binding to the cell surface MT1-MMP*TIMP-2 (membrane type-1 matrix metalloproteinase/tissue inhibitor of matrix metalloproteinase-2) complex with subsequent cleavage of proMMP-2 by TIMP-2-free adjacent MT1-MMP. This is followed by autolytic maturation of the activation intermediate and the release of the mature MMP-2 species from cell surfaces into the extracellular milieu. To observe the MMP-2 activation pathway in more detail, proMMP-2-deficient MCF7 breast carcinoma cells expressing MT1-MMP were incubated with excess proMMP-2 to saturate the available MT1-MMP*TIMP-2 surface receptors. After removal of the unbound material, the kinetics of proMMP-2 activation and MMP-2 release from cells into media was monitored by gelatin zymography and substrate cleavage. Our observations demonstrate that gelatin zymography is insufficient for providing meaningful information about the status of MMP-2. The proteolytically competent mature MMP-2 moiety alone, but not in its complex with TIMP-2, was released from the cells. In tissue culture conditions, the enzyme's proteolytic activity was suppressed in the next 30 to 60 minutes by tissue inhibitors of MMPs, especially by TIMP-1. The picture emerges that there is a likely temporal regulation of MMP-2 activity by TIMPs in tumor cells. These relatively rapid changes of the MMP-2 status cannot be detected by gelatin zymography. Additional studies are needed to examine the significance of this phenomenon in vivo.

Breast Neoplasms↗

PubMed2001

The hemopexin-like C-terminal domain of membrane type 1 matrix metalloproteinase regulates proteolysis of a multifunctional protein, gC1qR.

Matrix metalloproteinases (MMPs) including membrane type 1 MMP (MT1-MMP) can degrade extracellular matrix and cell surface receptor molecules and have an essential function in malignancy. Recently, we established a functional link between MT1-MMP and the receptor of complement component 1q (gC1qR). The gC1qR is known as a compartment-specific regulator of diverse cellular and viral proteins. Once released by proliferating cells, soluble gC1qR may inhibit complement component 1q hemolytic activity and play important roles in vivo in assisting tumor cells to evade destruction by complement. Here, we report that gC1qR is susceptible to MT1-MMP proteolysis in vitro and in cell cultures. The major MT1-MMP cleavage site (Gly(79) down arrow Gln(80)) is localized within the structurally disordered loop connecting the beta(3) and the beta(4) strands of gC1qR. The recombinant MT1-MMP construct that included the catalytic domain but lacked the hemopexin-like domain lost the proteolytic capacity; however, it retained the ability to bind gC1qR. Inhibition of MT1-MMP activity by a hydroxamate inhibitor converted the protease into a cell surface receptor of gC1qR and promoted co-precipitation MT1-MMP with the soluble gC1qR protein. It is tempting to hypothesize that these novel mechanisms may play important roles in vivo and have to be taken into account in designing hydroxamate-based cancer therapy.

Amino Acid Sequence↗

PubMed2001

An alternative processing of integrin alpha(v) subunit in tumor cells by membrane type-1 matrix metalloproteinase.

Membrane type-1 matrix metalloproteinase (MT1-MMP) and alpha(v)beta(3) integrin are both essential to cell invasion. Maturation of integrin pro-alpha(v)chain (pro-alpha(v)) involves its cleavage by proprotein convertases (PC) to form the disulfide-bonded 125-kDa heavy and 25-kDa light alpha chains. Our report presents evidence of an alternative pathway of pro-alpha(v) processing involving MT1-MMP. In breast carcinoma MCF7 cells deficient in MT1-MMP, pro-alpha(v) is processed by a conventional furin-like PC, and the mature alpha(v) integrin subunit is represented by the 125-kDa heavy chain and the 25-kDa light chain commencing from the N-terminal Asp(891). In contrast, in cells co-expressing alpha(v)beta(3) and MT1-MMP, MT1-MMP functions as an integrin convertase. MT1-MMP specifically cleaves pro-alpha(v), generating a 115-kDa heavy chain with the truncated C terminus and a 25-kDa light chain commencing from the N-terminal Leu(892). PC-cleavable alpha(3) and alpha(5) but not the PC-resistant alpha(2) integrin subunit are also susceptible to MT1-MMP cleavage. These novel mechanisms involved in the processing of integrin alpha subunits underscore the significance and complexity of interactions between MT1-MMP and adhesion receptors and suggest that regulation of integrin functionality may be an important role of MT1-MMP in migrating tumor cells.

Amino Acid Sequence↗

PubMed2001

Processing of integrin alpha(v) subunit by membrane type 1 matrix metalloproteinase stimulates migration of breast carcinoma cells on vitronectin and enhances tyrosine phosphorylation of focal adhesion kinase.

Recently, we have shown that membrane type 1 matrix metalloproteinase (MT1-MMP) exhibits integrin convertase activity. Similar to furin-like proprotein convertases, MT1-MMP directly processes a single chain precursor of alpha(v) integrin subunit (pro-alpha(v)) into the heavy and light alpha-chains connected by a disulfide bridge. To evaluate functionality of MT1-MMP-processed integrins, we examined breast carcinoma MCF7 cells co-expressing alpha(v)beta(3) integrin with either the wild type or mutant MT1-MMP in a variety of migration and adhesion tests. Specific inhibitors of proprotein convertases and MMP were employed in our cell system to attenuate the individual pathways of pro-alpha(v) maturation. We present evidence that MT1-MMP cleavage of pro-alpha(v) in the cells did not affect RGD-ligand binding of the resulting alpha(v)beta(3) integrin but enhanced outside-in signal transduction through a focal adhesion kinase pathway. Enhanced tyrosine phosphorylation of focal adhesion kinase in cells co-expressing MT1-MMP and alpha(v)beta(3) integrin contributed to efficient adhesion and, especially, migration of cells on vitronectin, a ligand of alpha(v)beta(3) integrin. These mechanisms underscore the significance of a coordinated interplay between MT1-MMP and alpha(v)beta(3) integrin in tumor cells and identify downstream signaling pathways resulting from their interactions. Regulation of integrin maturation and functionality may be an important role of MT1-MMP in tumor cells.

Antineoplastic Agents↗

PubMedDate not supplied

The matrix metalloproteinase-21 gene 572C/T polymorphism and the risk of breast cancer.

BACKGROUND: Matrix metalloproteinases (MMPs) contribute in multiple ways to all stages of tumor development, and a number of DNA polymorphisms in the MMP genes are associated with an increased risk of cancer. We previously identified the MMP-21 gene 572C/T polymorphism leading to Ala191Val substitution within the enzyme's catalytic domain. We performed a case-control study to test association between this polymorphism and the risk of breast cancer. PATIENTS AND METHODS: 572C/T polymorphism was analyzed by RFLP method in 396 unrelated Russian females: 76 breast cancer patients and 320 disease-free blood donors. RESULTS: The frequencies of C/C, T/C and T/T genotypes in patients (69.7%, 25.0% and 5.3%) did not differ significantly from those in controls (61.9%, 34.7% and 3.4%); the polymorphism was not associated with the increased tumor size and the presence of metastases. CONCLUSION: The MMP-21 gene 572C/T polymorphism has no significant effect on the development and progression of breast cancer.

Adolescent↗