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K Bein

Publications and source records attributed to K Bein.

12 recordsLinked to original sources
PubMed2000

Thrombospondin type 1 repeats interact with matrix metalloproteinase 2. Regulation of metalloproteinase activity.

Thrombospondins are thought to function as inhibitors of angiogenesis. However, the mechanism(s) of this activity is not well understood. In this study, we have used the yeast two-hybrid system to identify proteins that interact with the thrombospondins 1 (TSP1) and 2 (TSP2) properdin-like type 1 repeats (TSR). One of the proteins identified that interacted with both TSR was matrix metalloproteinase 2 (MMP2). The isolated MMP2 cDNA clone encoded amino acid residues 237-633, which include the fibronectin-like gelatin binding region flanking the catalytic center and the carboxyl hemopexin-like region. Further testing of this clone demonstrated that the TSR interacted with the NH(2)-terminal region of the MMP2 that contains the catalytic domain. The protein interaction observed in yeast was further demonstrated by immunoprecipitation and Western blotting using purified intact TSP1, TSP2, MMP2, and MMP9. Although MMP2 interacted with TSP1 and TSP2 via its gelatin-binding domain or a closely mapping site, neither TSP1 nor TSP2 was degraded by MMP2 in vitro. Tissue culture and in vitro assays demonstrated that the presence of purified TSR and intact TSP1 resulted in inhibition of MMP activity. The ability of TSP1 to inhibit MMP3-dependent activation of pro-MMP9 and thrombin-induced activation of pro-MMP2 suggests that the TSPs may inhibit MMP activity by preventing activation of the MMP2 and MMP9 zymogens.

Animals↗
PubMed1998

Myb-dependent regulation of thrombospondin 2 expression. Role of mRNA stability.

The nuclear transcription factor c-Myb, which is highly expressed in hematopoietic cells, has been shown to be functional in NIH 3T3 cells: cells that do not possess detectable levels of c-Myb. To identify endogenous target genes of c-Myb in fibroblasts, RNA isolated from NIH 3T3 cells stably transfected with a full-length or a dominant negative c-myb construct (GREMyb and GREMEn, respectively) was subjected to differential display analysis. 5'-Rapid amplification of cDNA ends of a selected band, sequencing, and a nucleotide homology search led to the identification of thrombospondin 2 (TSP 2) as the gene product repressed in GREMyb and induced in GREMEn cells. The pattern of TSP 2 expression during the cell cycle was consistent with c-myb-dependent regulation. The possibility that the identified transcript was TSP 1, a homologous product known to be repressed by v-Src, c-Jun, and v-Myc, was ruled out by using a TSP 2-specific DNA probe and by showing a distinct pattern of regulation of TSP 1 and TSP 2 expression. Nuclear run-on and TSP 2 promoter-reporter (chloramphenicol acetyltransferase) assays showed similar transcriptional levels in GREMyb and NIH 3T3 cells. However, mRNA stability studies showed a much shorter TSP 2 mRNA half-life in GREMyb compared with wild type NIH 3T3 cells, suggesting that c-myb affects TSP 2 expression via a post-transcriptional mechanism. The implications of a protooncogene-mediated suppression of TSP expression are discussed.

3T3 Cells↗
PubMed1997

c-Myb function in fibroblasts.

The protooncogene c-myb is a nuclear transcription factor that shares significant sequence homology with two other myb family members, A-myb and B-myb. Recent studies have suggested that c-myb is involved in regulation of the cell cycle via control of intracellular calcium [Ca2+]i concentration. Given the limited cell type expression of the c-myb gene, we set out to investigate whether myb-dependent cell cycle regulation occurs in cells not known to express the c-myb protein. NIH 3T3 fibroblasts were stably transfected with an inducible c-myb dominant negative construct composed of a myb DNA binding domain linked to the Drosophila engrailed transcription suppresser (pGREMEn) and a full-length murine c-myb cDNA sequence. Induced expression of the dominant negative construct was associated with a G1 cell cycle arrest and a failure to increase late G1 intracellular calcium levels. Similar expression studies in mouse embryonic fibroblasts derived from the c-myb knockout mouse have demonstrated lower baseline [Ca2+]i levels than in normal mice fibroblasts that were not further lowered by MEn expression. We conclude that regulation of calcium homeostasis and cell cycle progression via myb-dependent transcription may play an important role in cells not possessing detectable levels of c-myb protein.

3T3 Cells↗
PubMed1997

Regulation of vascular smooth muscle cell proliferation by plasma membrane Ca(2+)-ATPase.

We have previously shown that reductions in c-Myb-dependent transcription inhibit cell cycle progression and decrease intracellular Ca2+ concentrations in vascular smooth muscle cells (VSMC). We now report that these effects are largely mediated by a 4- to 10-fold increased rate of La(3+)-sensitive 45Ca extrusion, which is associated with 2- to 4-fold increased levels of plasma membrane Ca(2+)-ATPase 1 (PMCA1) mRNA and protein. PMCA4 mRNA, present at much lower concentrations, undergoes similar changes during suppression of c-Myb activity. We also report that PMCA1 expression is regulated during VSMC cell cycle progression, such that levels of PMCA1 are 40% lower at the G1/S interface than at G0. Moreover, transient overexpression of PMCA1a in VSMC elevates the 45Ca efflux rate by approximately 2-fold, decreases resting and peak thapsigargin-releasable Ca2+ concentrations at G1/S by 43% (68 nM) and 52% (160 nM), respectively, and reduces the rate of cell proliferation by over 2.5-fold. These data define a mechanism for c-Myb-dependent Ca2+ homeostasis and support a critical role for PMCA in the regulation of VSMC growth.

Animals↗
PubMed1997

c-Myb-dependent cell cycle progression and Ca2+ storage in cultured vascular smooth muscle cells.

Considerable controversy surrounds the role of the c-myb proto-oncogene in vascular smooth muscle cells (VSMCs). Previous investigations using antisense approaches have suggested a relationship between c-myb expression, cell cycle progression, and cytoplasmic Ca2+ concentration ([Ca2+]cyt). However, the ability of certain antisense oligonucleotides to bind and inactivate growth factors allows alternative explanations. To define more specifically the role of c-Myb in cultured VSMCs (SVE and A10 cell lines), we have generated stable cell clones expressing a dominant-negative c-Myb lacking critical elements of the DNA binding domain (delta5-SVE) and transiently transfected cell populations (GRE-MEn-SVE and GRE-MEn-A10) expressing a glucocorticoid-inducible chimeric protein that targets the Drosophila Engrailed repressor domain to c-Myb-responsive promoters. The delta5-SVE clones and GRE-MEn cell populations exhibit a 60% reduction in mean intracellular c-Myb activity, as measured by cotransfection assays with a c-Myb-responsive reporter, a 42% decrease in the mean S phase entry of growth-arrested (G[0]) cells after serum stimulation, and a 36% inhibition of mean cell proliferation over 4 days. These cells also display 28% (34-nmol/L) and 30% (42-nmol/L) reductions in mean [Ca2+]cyt at G(0) and at the G1/S interface, respectively, as well as significant reductions in the peak [Ca2+]cyt responses to thapsigargin (5 micromol/L) and caffeine (10 mmol/L). These latter reductions in operationally defined Ca2+ pools were observed both at different stages of the cell cycle and after transient induction of the dominant-interfering construct, suggesting that c-Myb regulates these releasable Ca2+ stores independent of its effects on cell cycle progression.

Animals↗
PubMed1991

Molecular cloning of a cDNA encoding the amino end of the mammalian multifunctional protein CAD and analysis of the 5'-flanking region of the CAD gene.

CAD is a 243-kDa multidomain polypeptide which catalyzes the first three steps in mammalian de novo pyrimidine biosynthesis. The largest cDNA clone obtained thus far, pCAD142 (Shigesada, K., Stark, G.R., Maley, J. A., Niswander, L. A., and Davidson, J. N. (1985) Mol. Cell. Biol. 5, 1735), lacks the 5' end of the mRNA which encodes the amino terminus of CAD. To clone this missing segment, a synthetic oligonucleotide complementary to pCAD142 and poly(A)+ RNA template, isolated from a Syrian hamster cell line which overproduces the CAD mRNA, were used for cDNA synthesis. The resulting clone pKB11, which has a 1369-base pair (bp) cDNA insert, overlapping pCAD142 by 781 bp, was identified by hybridization methods and sequence analysis and found to contain the entire cDNA sequence for the amino end of the CAD polypeptide. The deduced amino acid sequence is homologous to seven carbamyl phosphate synthetases. Primer extension, oligonucleotide-directed RNase H digestion, and RNA sequencing indicated that pKB11 extends to within 68 bases of the 5' end of the CAD mRNA. This conclusion was confirmed by Northern blotting analysis of the 5'-flanking region of CAD gene. The probable 3' end of an unidentified gene which codes for a 1-kilobase (kb) transcript was identified immediately upstream of the CAD gene. Northern analysis using probes complementary to the region between the CAD and the 1-kb genes detected the presence of a small transcript of less than 300 nucleotides. The sequence revealed three potential translation initiation sites raising the possibility of more than one CAD translation product. The major translation start codon was identified as the first ATG in pKB11 by sequence homology, in vitro transcription and translation, and protein studies. Starting from this ATG within pKB11, the clone encodes a 143-residue domain of unknown function. This study completes the determination of the primary structure of the CAD polypeptide. The CAD mRNA is 7.5 kb in length and has 6675 bp of coding sequence and about 200 bp and 600 bp of untranslated sequence at the 5' and 3' ends, respectively.

Amino Acid Sequence↗
PubMed1967

School phobia.

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