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S Zucker

Publications and source records attributed to S Zucker.

At least 37 records · Page 2Linked to original sources
PubMed1999

Developmental expression of the protein kinase C substrate/binding protein (clone 72/SSeCKS) in rat testis identification as a scaffolding protein containing an A-kinase-anchoring domain which is expressed during late-stage spermatogenesis.

The coordinated interaction of kinases, phosphatases and other regulatory molecules with scaffolding proteins is emerging as a major theme in intracellular signaling networks. In this report we show that a cDNA isolated from a rat testis expression library by interactive cloning using the regulatory subunit (R) of a type-II protein kinase A (PKA) is identical with a previously characterized protein kinase C (PKC)-binding protein termed either clone 72 [Chapline, C., Mousseau, B., Ramsay, K., Duddy, S., Li, Y., Kiley, S. C. & Jaken, S. (1996) J. Biol. Chem. 271, 6417-6422] or SSeCKS [Lin, X., Tombler, E., B., Nelson, P.J., Ross, M. & Gelman, I.H. (1996) J. Biol. Chem. 271, 28430-28438]. Deletion mutagenesis demonstrated that amino acids 1495-1524 of clone 72/SSeCKS had the ability to interact with RII. Antibodies prepared against the recombinant protein recognized a 280/290-kDa doublet and a 240-kDa protein on Western blots of rat testis cytosolic and Triton X-100 extracts. Expression of clone 72/SSeCKS mRNA and protein levels was developmentally regulated in rat testis. Northern-blot analysis showed a dramatic increase in clone 72/SSeCKS-hybridizing mRNA starting 30 days after birth. Immunohistochemical examination showed high expression levels in elongating spermatids. Clone 72/SSeCKS was not detected in mature sperm. These studies suggest a role for clone 72/SSeCKS, a PKA/PKC scaffolding protein, during the process of spermiogenesis.

A Kinase Anchor Proteins↗
PubMed1999

Regulation of gelatinases in human airway smooth muscle cells: mechanism of progelatinase A activation.

Matrix metalloproteinases (MMPs) play an important role in tumor metastasis and invasion, inflammatory tissue destruction and remodeling, wound healing, and angiogenesis. The 72-kDa gelatinase A is the most widely distributed of all the MMPs, and along with the 92-kDa gelatinase B, both play an important role in the turnover of basement membrane. The role of MMPs in chronic airway inflammation and remodeling has received scant attention. In this study, we sought to examine the release and activation of gelatinases from human airway smooth muscle (ASM) cells and the effect of tumor necrosis factor-alpha and phorbol 12-myristate 13-acetate (PMA) on this release and activation. The role of membrane type 1 MMP (MT1-MMP) and tissue inhibitor of MMP (TIMP)-2 in activating progelatinase A has been explored. We have demonstrated, using human airway smooth muscle cells in culture, that 1) ASM releases gelatinase A constitutively and when stimulated with PMA and tumor necrosis factor-alpha releases gelatinase B, and the release of gelatinase B is protein kinase C dependent because it is blocked by H-7 and staurosporin; 2) treatment of ASM cells with PMA or concanavalin A failed to activate progelatinase A despite these agents increasing cell expression of MT1-MMP; and 3) the inability of ASM cell membranes to activate progelatinase A is most likely secondary to the high levels of TIMP-2 on the cell membrane. In conclusion, our results demonstrate that human ASM cells constitutively secrete progelatinase A and when stimulated with proinflammatory mediators secrete gelatinase B. The released gelatinases A and B may be important factors in the airway remodeling that occurs in asthma.

Cell Membrane↗
PubMed1999

Expression of the extracellular matrix metalloproteinase inducer (EMMPRIN) and the matrix metalloproteinase-2 in bronchopulmonary and breast lesions.

Tumor cells interact with stromal cells via soluble or cell-bound factors stimulating the production of matrix metalloproteinases (MMPs), a group of enzymes largely involved in the extracellular matrix (ECM) remodeling in tumor invasion. Among these factors, extracellular matrix metalloproteinase inducer (EMMPRIN) has been shown to stimulate in vitro the fibroblast production of various MMPs such as interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), and gelatinase A (MMP-2). In this study, the EMMPRIN protein was detected by immunohistochemistry prominently in malignant proliferations of the breast and the lung. It was present at the surface of both tumor epithelial and peritumor stromal cells. Because previous studies have reported that stromal cells do not express EMMPRIN mRNAs, it is very likely that EMMPRIN is bound to stromal cells via a specific receptor. Moreover, our observations also demonstrated that the same peritumor stromal cells strongly express MMP-2. Our results show that EMMPRIN is an important factor in tumor progression by causing tumor-associated stromal cells to increase their MMP-2 production, thus facilitating tumor invasion and neoangiogenesis. (J Histochem Cytochem 47: 1575-1580, 1999)

Adenocarcinoma↗
PubMed1999

Increased serum stromelysin-1 levels in systemic lupus erythematosus: lack of correlation with disease activity.

OBJECTIVE: In view of evidence that stromelysin-1 and collagenase-1 are involved in tissue injury in inflammatory joint diseases, we sought to determine whether matrix metalloproteinases (MMP) are implicated in the pathophysiology of systemic lupus erythematosus (SLE). METHODS: Seventy-three patients with SLE and 39 healthy subjects were evaluated. Serum levels of MMP and tissue inhibitor of metalloproteinases were measured. RESULTS: Serum stromelysin-1 levels were significantly increased in patients with SLE (416+/-252 ng/ml) compared to healthy subjects (125+/-93 ng/ml). No correlation between serial measurements of stromelysin-1 and disease activity in SLE patients was noted. Serum collagenase-1, gelatinase A, and TIMP-1 levels were not increased in SLE. CONCLUSION: Serum concentrations of stromelysin-1 are increased in SLE, but the levels do not correlate with disease activity.

Disease Progression↗
PubMed1998

The propeptide domain of membrane type 1 matrix metalloproteinase is required for binding of tissue inhibitor of metalloproteinases and for activation of pro-gelatinase A.

Activation of secreted latent matrix metalloproteinases (MMPs) is accompanied by cleavage of the N-terminal propeptide, thereby liberating the active zinc from binding to the conserved cysteine in the pro-domain. It has been assumed that an analogous mechanism is responsible for the activation of membrane type 1 MMP (MT1-MMP). Using recombinant wild-type MT1-MMP cDNA and mutant cDNAs transfected into COS-1 cells lacking endogenous MT1-MMP, we have examined the function of the propeptide domain of MT1-MMP. MT1-MMP was characterized by immunoblotting, surface biotinylation, gelatin substrate zymography, and 125I-tissue inhibitor of metalloproteinases 2 (TIMP-2) binding. In contrast to wild-type MT1-MMP-transfected COS-1 cells, transfected COS-1 cells containing a deletion of the N-terminal propeptide domain of MT1-MMP or a chimeric construction (substitution of the pro-domain of MT1-MMP with that of collagenase 3) were functionally inactive in terms of binding of 125I-labeled TIMP-2 to the cell surface and initiating the activation of pro-gelatinase A. These results support the concept that in its native plasma membrane-inserted form, the pro-domain of MT1-MMP plays an essential role in TIMP-2 binding and subsequent activation of pro-gelatinase A.

Enzyme Activation↗
PubMed1998

Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation.

Production of vascular endothelial growth factor (VEGF) by cancer cells at invasive and metastatic sites is an important aspect of tumor angiogenesis. Although known primarily as a mitogen and a vascular permeability factor (VPF) for endothelial cells, VEGF/VPF has been proposed to induce the expression of procoagulant factors in endothelial cells. In this study, we have explored the ramifications of VEGF induction of tissue factor (TF) in human umbilical vein endothelial cells (HUVECs) and subsequent activation of progelatinase A. Within 3 hr of incubation with VEGF/VPF, endothelial cells accelerate TF generation as measured using chromogenic substrate assays for coagulation factors Xa and thrombin. Incubation of VEGF/VPF-pre-treated cells with prothrombin and factors X, Va, and VIIa at 37 degrees C and subsequent generation of thrombin resulted in activation of secreted endothelial progelatinase A as demonstrated by gelatin zymography. Anti-thrombin III or antibodies to TF inhibited thrombin generation and progelatinase A activation. VEGF/VPF also directly increased HUVEC secretion of interstitial collagenase, tissue inhibitor of metalloproteinases (TIMP-1) and, to a lesser extent, gelatinase A. The effect of thrombin on endothelial proliferation in serum-free media was examined. Thrombin was a growth factor for HUVECs at a lower dose than that required for progelatinase A activation. Whereas TIMP-2 abrogated thrombin-induced progelatinase A activation, it had no significant effect on thrombin-induced endothelial cell growth. We propose that an early step in tumor angiogenesis involves VEGF-induced thrombin generation and increased MMP production with subsequent activation of endothelial progelatinase A and degradation of the underlying basement membrane.

Cell Division↗
PubMed1998

Tissue inhibitor of metalloproteinase-2 (TIMP-2) binds to the catalytic domain of the cell surface receptor, membrane type 1-matrix metalloproteinase 1 (MT1-MMP).

It has been proposed that tissue inhibitor of metalloproteinase-2 (TIMP-2), in stoichiometric concentrations, serves as an intermediate in progelatinase A activation by binding to activated membrane type 1-matrix metalloproteinase 1 (MT1-MMP) on the plasma membrane. An MT1-MMP-independent cell surface receptor for TIMP-2 has also been postulated. To clarify TIMP-2 binding, we have performed 125I-TIMP-2 binding studies on transfected COS-1 cells and endothelial cells. Specific receptors for TIMP-2 were identified on COS-1 cells transfected with MT1-MMP cDNA, but not on vector-transfected cells. Treatment of MT1-MMP transfected COS-1 cells with a hydroxamic acid inhibitor of MMPs, CT-1746, but not an inactive stereoisomer, CT-1915, produced dose-dependent inhibition of specific TIMP-2 binding comparable with that noted with excess unlabeled TIMP-2. This result suggests that TIMP-2 binds to the zinc catalytic site of MT1-MMP. As demonstrated by the limited competition for binding of C-terminal deleted TIMP-2, the C-terminal domain of TIMP-2 participates in binding to MT1-MMP. Cross-linking studies followed by immunoprecipitation using antibodies to MT1-MMP were employed to identify 125I-TIMP-2.MT1-MMP complexes in MT1-MMP-transfected COS-1 cell membrane extracts. TIMP-2 receptors were also identified on concanavalin A-treated human umbilical vein endothelial cells; inhibition of TIMP-2 binding with CT-1746 was demonstrated.

Amino Acid Sequence↗
PubMed1998

Instructional intranets in graduate medical education.

Changes in medicine, medical education, and technology have influenced graduate medical education (GME) and have altered many traditional concepts of resident training. Three issues in particular have led to changes. The first is the shortage of time that academic and community physicians have to devote to medical teaching because of the demands to bring in revenue through clinical practice. The second is the limited exposure that residents have to various medical conditions due to a shift in training venues from hospitals to ambulatory care settings. Last is residents' lack of training in using information technologies. The resultant deficits the exist in GME make it more difficult for residents to practice medicine in the most efficient manner. Hence, there is a need for health care professionals' education to address the coming demands of the 21st century. Instructional computer technology can be useful in bridging this gap. Intranets, internal organizational networks, are private versions of the World Wide Web that are often available only to members of a particular organization. This paper reviews changes in medicine and medical education, describes how instructional intranets can be incorporated into GME, and discusses the impact intranet and Internet technologies can have on GME.

Computer Communication Networks↗
PubMed1997

Stimulation of matrix metalloproteinase production by recombinant extracellular matrix metalloproteinase inducer from transfected Chinese hamster ovary cells.

Many of the tumor-associated matrix metalloproteinases that are implicated in metastasis are produced by stromal fibroblasts within or surrounding the tumor in response to stimulation by factors produced by tumor cells. In this study we transfected Chinese hamster ovary cells with putative cDNA for human extracellular matrix metalloproteinase inducer (EMMPRIN), a transmembrane glycoprotein that is attached to the surface of many types of malignant human tumor cells and that has previously been implicated in stimulation of matrix metalloproteinase production in fibroblasts. We show that these transfected cells synthesize EMMPRIN that is extensively post-translationally processed; this recombinant EMMPRIN stimulates human fibroblast production of interstitial collagenase, stromelysin-1, and gelatinase A (72-kDa type IV collagenase). We propose that EMMPRIN regulates matrix metalloproteinase production during tumor invasion and other processes involving tissue remodeling.

Animals↗
PubMed1997

Tumor collagenase stimulatory factor (TCSF) expression and localization in human lung and breast cancers.

Tumor cell-derived collagenase stimulatory factor (TCSF) stimulates in vitro the biosynthesis of various matrix metalloproteinases involved in tumor invasion, such as interstitial collagenase, gelatinase A, and stromelysin 1. The expression of TCSF mRNAs was studied in vivo, using in situ hybridization and Northern blotting analysis, in seven normal tissues and in 22 squamous cell carcinomas of the lung, and in seven benign proliferations and in 22 ductal carcinomas of the mammary gland. By in situ hybridization, TCSF mRNAs were detected in 40 of 44 carcinomas, in pre-invasive and invasive cancer cells of both lung and breast cancers. TCSF mRNAs and gelatinase A mRNAs were both visualized in the same areas in serial sections in breast cancers, and were expressed by different cells, tumor cells, and fibroblasts. The histological results were confirmed by Northern blot analysis, which showed a higher expression of TCSF mRNAs in cancers than in benign and normal tissues. These observations support the hypothesis that TCSF is an important factor in lung and breast tumor progression.

Antigens, CD↗
PubMed1996

Membrane type matrix metalloproteinase 1 activates pro-gelatinase A without furin cleavage of the N-terminal domain.

Membrane type matrix metalloproteinase 1 (MT-MMP1), a novel 63-kDa member of the matrix metalloproteinase family, is a membrane-anchored enzyme and an activator for gelatinase A. In addition to its C-terminal hydrophobic transmembrane domain, MT-MMP1 has an insertion of 11 amino acids between its propeptide and catalytic domain encrypted with a RRKR recognition motif for the paired basic amino acid cleaving enzyme, furin. In this report, we investigated whether the cleavage of the RRKR motif of MT-MMP1 by Golgi-associated furin is analogous to a similar enzyme activation mechanism observed with stromelysin-3. Mutant forms of MT-MMP1 were cotransfected into COS-1 cells with cDNAs for pro-gelatinase A and/or furin. Immunoprecipitation and immunoblotting using specific antibodies were employed to characterize cell proteins. Whereas furin readily cleaved soluble MT-MMP1 lacking the transmembrane domain (DeltaMT-MMP1), a soluble stromelysin-1/DeltaMT-MMP1 chimera without the RRKR basic motif was resistant to furin-induced cleavage. COS-1 cells cotransfected with wild type MT-MMP1 cDNA and furin cDNA demonstrated a 63-kDa protein (latent enzyme) on SDS-polyacrylamide gel electrophoresis rather than the anticipated lower molecular weight activated enzyme. Inhibition of furin activity with alpha1-protease inhibitorPittsburgh (a furin inhibitor) did not affect the pro-gelatinase A activation mechanism in COS-1 cells cotransfected with MT-MMP1 and pro-gelatinase A cDNAs. Furthermore, substitution of the RRKR motif of MT-MMP1 with alanine residues by site-directed mutagenesis resulted in the same 63-kDa protein without loss of pro-gelatinase A activation function. These data indicate that furin-induced activation of MT-MMP1 is not a prerequisite for pro-gelatinase A activation. The mechanism of activation of cell-bound MT-MMP1 remains to be elucidated.

Animals↗
PubMed1996

Activation of human umbilical vein endothelial cell progelatinase A by phorbol myristate acetate: a protein kinase C-dependent mechanism involving a membrane-type matrix metalloproteinase.

Matrix metalloproteinases play an important role in tumor invasion, angiogenesis and inflammatory tissue destruction. The 72-kd gelatinase A is the most widely distributed. Along with the 92-kd gelatinase B, it plays an important role in basement membrane turnover. Gelatinase A is secreted as progelatinase A and, when activated, can cause extracellular matrix destruction. The physiologic mechanism of this activation is not well understood. Based on the importance of endothelial cells in inflammation and cancer, we sought in this study to systematically study the PMA-induced activation of endothelial cell progelatinase A. Using HUVEC, we demonstrated that PMA-induced activation of progelatinase A in these vascular endothelial cells (a) was protein kinase C-dependent as it was blocked by H-7; (b) occurred through cell-mediated events as PMA was unable to activate progelatinase A in a cell-free system and that low dose tissue inhibitor of metalloproteinases-2, but not tissue inhibitor of metalloproteinases-1, totally inhibited PMA-induced activation; (c) was accompanied by an increase in the membrane-type matrix metalloproteinase (MT-MMP). We also found that the combination of PMA and the cytokine tumor necrosis factor-alpha increased HUVEC secretion and activation of gelatinase B. In conclusion, our data show that PMA activation of vascular endothelial cell progelatinase A is a cell membrane event that is at least partially mediated through a PKC-dependent mechanism and is accompanied by an increase synthesis of MT-MMP. These data suggest a role for MT-MMP in the activation of progelatinase A in vascular endothelial cells.

Cell Membrane↗
PubMed1995

Thrombin induces the activation of progelatinase A in vascular endothelial cells. Physiologic regulation of angiogenesis.

Angiogenesis requires degradation of vascular basement membrane prior to migration and proliferation of endothelial cells; proteinases are essential ingredients in this process. Because of thrombin's multiple effects on endothelium, we have examined its role in matrix metalloproteinase activation using human umbilical vein endothelial cells. Gelatin zymography of endothelial conditioned media revealed a prominent 72-kDa progelatinase A band. Addition of alpha-thrombin to endothelial cells resulted in the generation of 64 and 62 kDa gelatinolytic bands which is consistent with the activation of progelatinase A; thrombin had no effect in the absence of cells. This effect requires the proteolytic site of thrombin since progelatinase A activation was abolished by specific inhibitors of thrombin. Matrix metaloproteinase inhibitors diminished thrombin-induced activation of progelatinase A. Pretreatment of endothelial cells with excess tissue inhibitor of metalloproteinase-2 or a COOH-terminal fragment of progelatinase A abrogated thrombin-mediated activation of progelatinase A presumably by competing with the COOH terminus of native progelatinase A for interaction with an activator site on endothelial plasma membranes. Although membrane-type matrix metalloproteinase was demonstrated in endothelial cells by Northern and Western blotting, the receptor function of this molecule in thrombin-induced activation of progelatinase A needs to be clarified. Progelatinase A activation did not require intracellular signal transduction events mediated by the thrombin receptor. These data demonstrate that 1) endothelial cells express a novel activation mechanism for progelatinase A, 2) proteolytically active thrombin regulates this activation mechanism, and 3) activation occurs independently of the functional thrombin receptor.

Amino Acid Chloromethyl Ketones↗
PubMed1995

Plasma assay of gelatinase B: tissue inhibitor of metalloproteinase complexes in cancer.

BACKGROUND: Matrix metalloproteinases (MMPs), especially gelatinase A and gelatinase B (GLB), are believed to be important components of the metastatic process. Tissue Inhibitors of Metalloproteinases (TIMPs) form complexes with MMPs and inhibit cancer dissemination. After local secretion, MMPs and their complexes with TIMPs leach into the blood stream where their concentration can be measured, thereby serving as surrogate markers of disease. Elevated plasma gelatinase B levels have been detected in gastrointestinal cancer and breast cancer. The goal of this study was to determine whether plasma GLB:TIMP complexes also are increased in cancer and whether these tests have potential use as prognostic tumor markers. METHODS: An enzyme-linked immunosorbent assay (ELISA) was developed to measure the plasma concentration of GLB:TIMP complexes in patients with cancer. Correlation between ELISA results and clinical outcome was sought. RESULTS: Plasma GLB:TIMP complexes were significantly increased in patients with gastrointestinal cancer and gynecologic cancer, but not in patients with breast cancer. When results from plasma GLB:TIMP complexes and plasma GLB assays were combined (GLB/complexes), abnormal levels of one or both assays were found in 36% and 65% of patients with gastrointestinal and gynecologic cancer, respectively. In Stage IV gastrointestinal cancer, patient survival was shorter (P < 0.001) in the group with increased plasma GLB/complexes than for those with normal plasma levels (4 months vs. 20 months, respectively). CONCLUSIONS: The assay of plasma gelatinase B and GLB:TIMP complexes may be clinically useful in predicting survival in subsets of patients with cancer. The possibility of using these assays in early stage cancer to predict metastasis should be studied.

Biomarkers, Tumor↗
PubMed1995

Localization of tissue inhibitor of matrix metalloproteinases in Alzheimer's disease and normal brain.

Based upon the hypothesis that metalloproteinases and their inhibitors might be involved in the pathogenesis of Alzheimer's disease, we studied brain samples of eight cases of Alzheimer's disease, six other pathological entities and three elderly controls for tissue inhibitor of metalloproteinase (TIMP) immunoreactivity. Specificity was supported by a loss of immunoreactivity following antigen preabsorption of antisera. Areas studied included ependyma, choroid plexus, frontoparietal, hippocampal and cerebellar cortex, n. basalis of Meynert, basal ganglia, midbrain, pons, and medulla. TIMP positivity was localized to neuritic senile plaques, neurofibrillary tangles and Purkinje cells. The pattern of TIMP plaque staining was similar to that observed with anti tau and SP18 antibodies. It differed from that observed with anti SP40, HAM 56 and GFAP antibodies. The selective localization of TIMP to the neuritic lesions of Alzheimer's disease in a codistribution with the amyloid precursor protein and abnormally phosphorylated and truncated tau supports a possible role for metalloproteinases and their inhibitors in the evolution of these lesions.

Aged↗