Biomedical subjects

Jorma Keski-Oja

Publications and source records attributed to Jorma Keski-Oja.

28 records · Page 2Linked to original sources

PubMed2003

Latent transforming growth factor-beta-binding protein 2 is an adhesion protein for melanoma cells.

Of the four latent transforming growth factor (TGF)-beta-binding proteins (LTBPs), LTBP-2 is different in the respect that it does not bind small latent forms of TGF-beta. LTBP-2 is therefore likely to have other roles in the extracellular matrix. LTBP-2 contains an RGD putative integrin recognition site, suggesting a role in cell adhesion. We carried out a study on cell attachment to LTBP-2. Purified recombinant LTBP-2 was used as substratum in cell adhesion and migration studies. We found that, unlike most adherent cell lines, all of the melanoma cell lines tested adhered to LTBP-2 very efficiently and in a concentration-dependent manner. Bowes melanoma cells bound most efficiently to LTBP-2 and were used for further characterization. Cell adhesion assays with recombinant LTBP-2 fragments indicated that the adhesive site is located in an N-terminal region of LTBP-2. The attachment of melanoma cells to LTBP-2 was prevented with monoclonal antibody against beta1 integrin in a concentration-dependent manner, suggesting an important role for beta1 integrin in the process. Antibodies against integrin subunits alpha3 and alpha6 decreased melanoma cell adhesion as well. The beta1 and alpha3 integrins were localized on the cell surface, especially in lamellipodia, as observed by immunofluorescence. In addition to integrin antagonists, heparin also markedly decreased melanoma cell adhesion. LTBP-2 also supported Bowes cell migration in modified Boyden chamber assays in a manner similar to the migration on fibronectin. Current data indicate that LTBP-2 can play a role in melanoma cell adhesion.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Matrix reloaded to circulation hits the tumor target.

Proteolytic fragments of various components of the extracellular matrix exhibit antiangiogenic activity via interaction with endothelial cell surface integrins. Kalluri and coworkers (this issue of Cancer Cell) use gene-targeted mice to show a physiological role for a carboxy-terminal fragment of collagen IV in the regulation of tumor angiogenesis.

Animals↗

PubMed2003

Expression of latent TGF-beta binding protein LTBP-1 is hormonally regulated in normal and transformed human lung fibroblasts.

Latent transforming growth factor-beta binding proteins (LTBPs), participate in the secretion, targeting and activation of transforming growth factor-beta (TGF-beta) isoforms. Besides the regulation of the bioavailability of TGF-betas, LTBPs are secreted as free forms and they associate with extracellular fibrillar structures. The current study was carried out to understand hormonal regulation of LTBP-1 expression in normal and SV-40 virus transformed human lung fibroblasts. LTBP-1 protein and mRNA expression were analysed by immunoblotting and Northern hybridisation. Basal expression levels of LTBP-1 were significantly reduced in the transformed cells. Retinoic acid (RA) as well as 9-cis-RA increased LTBP-1 expression in both cell lines, probably through post-transcriptional mechanisms. Dexamethasone induced LTBP-1 protein and mRNA levels in both cell lines. The effect was much more prominent in transformed cells and involved both transcriptional and post-transcriptional events. Vitamin D, estradiol and tamoxifen had only negligible effects on LTBP-1 expression. TGF-beta1 increased its own expression as well as LTBP-1 levels in both cell lines, but the response was clearly of lower magnitude in transformed cells. This may reflect insensitivity to TGF-beta1, which is a common feature for malignant cells. Our results indicate that LTBP-1 expression is under the regulation of RA and corticoids in lung fibroblasts. The responses to these hormonal modulators may either be lost or enhanced in transformed cells, which likely contributes to malignant growth.

Adrenal Cortex Hormones↗

PubMed2002

Endostatin associates with integrin alpha5beta1 and caveolin-1, and activates Src via a tyrosyl phosphatase-dependent pathway in human endothelial cells.

Endostatin, the COOH-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis and tumor growth. To understand the mechanisms behind endostatin action, we analyzed the plasma membrane- extracellular matrix interactions of recombinant human endostatin in cultured microvascular endothelial cells. We observed that endostatin induced rapid clustering of alpha5beta1 integrin associated with actin stress fibers and its concomitant colocalization with the membrane anchor protein caveolin-1. Furthermore, endostatin could be coimmunoprecipitated with alpha5beta1 and caveolin-1 from endothelial cell extracts. Endostatin treatment induced phosphatase-dependent activation of caveolin-associated Src family kinases. The disassembly of actin stress fibers and focal adhesions by endostatin was found to occur via activation of Src and in a tyrosyl phosphatase-dependent manner. The endostatin-treated cells void of the focal adhesions had impaired ability to deposit fibronectin into their extracellular matrices and were unable to migrate in response to basic fibroblast growth factor in a wounding experiment. These results indicate that recombinant endostatin interacts with alpha5beta1 integrin and caveolin-1 at the endothelial cell surface. In addition, the antimigratory effect of endostatin involves phosphatase-dependent Src activation and impaired cell-matrix interactions.

Actins↗

PubMed2002

Disruption of the gene encoding the latent transforming growth factor-beta binding protein 4 (LTBP-4) causes abnormal lung development, cardiomyopathy, and colorectal cancer.

Transforming growth factor-betas (TGF-betas) are multifunctional growth factors that are secreted as inactive (latent) precursors in large protein complexes. These complexes include the latency-associated propeptide (LAP) and a latent transforming growth factor-beta binding protein (LTBP). Four isoforms of LTBPs (LTBP-1-LTBP-4) have been cloned and are believed to be structural components of connective tissue microfibrils and local regulators of TGF-beta tissue deposition and signaling. By using a gene trap strategy that selects for integrations into genes induced transiently during early mouse development, we have disrupted the mouse homolog of the human LTBP-4 gene. Mice homozygous for the disrupted allele develop severe pulmonary emphysema, cardiomyopathy, and colorectal cancer. These highly tissue-specific abnormalities are associated with profound defects in the elastic fiber structure and with a reduced deposition of TGF-beta in the extracellular space. As a consequence, epithelial cells have reduced levels of phosphorylated Smad2 proteins, overexpress c-myc, and undergo uncontrolled proliferation. This phenotype supports the predicted dual role of LTBP-4 as a structural component of the extracellular matrix and as a local regulator of TGF-beta tissue deposition and signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Secretion of human latent TGF-beta-binding protein-3 (LTBP-3) is dependent on co-expression of TGF-beta.

Latent TGF-beta-binding proteins (LTBPs) were initially identified through their binding to the growth factor. Three of the four known LTBPs are able to associate covalently with the small latent forms of TGF-beta and mediate their efficient secretion. LTBPs have subsequently been found to associate with the extracellular matrix. We report here the cDNA cloning and characterization of the human LTBP-3 protein, which is the smallest LTBP. The hLTBP-3 gene consists of 28 exons, including one alternatively spliced exon. The splice variant contains an additional epidermal-growth-factor-like repeat in the C-terminus. The gene is transcribed to produce a approximately 4.6 kb mRNA, which is expressed at high levels in human heart, skeletal muscle, prostate and ovaries and in certain osteosarcoma and fibroblastic cell lines. Antibodies were generated against recombinant fragment of hLTBP-3 and used to detect the protein and its secretion from cultured COS-7 and osteosarcoma cells. Immunoblotting analysis indicated that efficient secretion of overexpressed hLTBP-3 from COS-7 cells required co-expression of TGF-beta1, which resulted in the secretion of high molecular weight complexes of approximately 240 kDa. hLTBP-3 protein was secreted from cultured osteosarcoma cells as high molecular weight complexes rather than in the free form. Similar complexes were recognized with antibodies specific to beta1*LAP. These findings indicate that human LTBP-3 has an essential role in the secretion and targeting of TGF-beta1.

Alternative Splicing↗

PubMed2002

Oligomerization through hemopexin and cytoplasmic domains regulates the activity and turnover of membrane-type 1 matrix metalloproteinase.

The formation of multimeric complexes by membrane-type 1 matrix metalloproteinase (MT1-MMP) may facilitate its autocatalytic inactivation or proMMP-2 activation on the cell surface. To characterize these processes, we expressed various glutathione S-transferase/MT1-MMP fusion proteins in human HT-1080 fibrosarcoma cells and SV40-transformed lung fibroblasts and analyzed their effects on MT1-MMP activity and potential homophilic interactions. We report here that MT1-MMP is expressed on the cell surface as oligomeric 200--240-kDa complexes containing both the active 60-kDa and autocatalytically processed 43-kDa species. Overexpression of a glutathione S-transferase/MT1-MMP fusion protein containing the transmembrane and cytoplasmic domains of MT1-MMP inhibited the phorbol 12-myristate 13-acetate-induced autocatalytic cleavage of endogenous MT1-MMP to the 43-kDa species, but not proMMP-2 activation. On the other hand, a similar fusion protein with the hemopexin, transmembrane, and cytoplasmic domains inhibited proMMP-2 activation in a dominant-negative fashion. These results suggest that both the autocatalytic cleavage of MT1-MMP and proMMP-2 activation may be regulated by oligomerization through the cytoplasmic and hemopexin domains. Indeed, either domain, when attached to the cell membrane by a transmembrane domain, formed stable homophilic complexes. Copurification of MT1-MMP with these fusion proteins correlated with their cell-surface co-localization. Thus, MT1-MMP oligomerization through the hemopexin, transmembrane, and cytoplasmic domains controls its catalytic activity.

Binding, Competitive↗

PubMed2002

Plasmin activates pro-matrix metalloproteinase-2 with a membrane-type 1 matrix metalloproteinase-dependent mechanism.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) has been implicated as a physiological activator of progelatinase A (MMP-2). We previously reported that plasmin treatment of cells results in proMMP-2 activation and increased type IV collagen degradation. Here, we analyzed the role of MT1-MMP in plasmin activation of MMP-2 using HT-1080 cells transfected with MT1-MMP sense or antisense cDNA. Control, vector-transfected cells that expressed endogenous MT1-MMP, and antisense cDNA transfectants with very low levels of MT1-MMP did not activate proMMP-2. Conversely, cells transfected with sense MT1-MMP cDNA expressed high MT1-MMP levels and processed proMMP-2 to 68/66-kDa intermediate activation products. Control cells and MT1-MMP transfectants had much higher levels of cell-associated MMP-2 than antisense cDNA transfectants. Addition of plasmin(ogen) to control or MT1-MMP-transfected cells generated active, 62-kDa MMP-2, but was ineffective with antisense cDNA transfectants. The effect of plasmin(ogen) was prevented by inhibitors of plasmin, but not by metalloproteinase inhibitors, implicating plasmin as a mechanism for proMMP-2 activation independent of the activity of MT1-MMP or other MMPs. Plasmin-mediated activation of proMMP-2 did not result from processing of proMT1-MMP and did not correlate with alpha(v)beta(3) integrin or TIMP-2 levels. Thus, plasmin can activate proMMP-2 only in the presence of MT1-MMP; however, this process does not require the catalytic activity of MT1-MMP.

DNA, Complementary↗

PubMed2002

Epilysin (MMP-28) expression is associated with cell proliferation during epithelial repair.

Epilysin (MMP-28) is the newest member of the matrix metalloproteinase enzyme family. Several members of this enzyme family have been associated with various aspects of wound repair and cancer invasion. The aim of this study was to characterize in different types of wounds, skin cancers, and keratinocyte cultures factors that contribute to epilysin expression in vivo, as well as how and where it is induced in relation to other matrix metalloproteinases. Our results indicate that epilysin is produced by the mitotic Ki-67-positive keratinocytes distal from the wound edge in both acute and chronic wounds and that it does not generally colocalize with collagenase-1, stromelysin-2, or 92 kDa gelatinase in migrating keratinocytes. An injury of epidermis was needed for epilysin induction as it was upregulated in ulcerated pyogenic granulomas and in suction blisters but was not detected in intact acanthotic or normal skin. Unlike many other matrix metalloproteinases, epilysin was not detected in the invading cancer cell nests of sclerosing basal or squamous cell cancers of various grades. When primary keratinocytes were stimulated with tumor necrosis factor alpha, upregulation of epilysin mRNA was evident within 24-48 h as measured by quantitative reverse transcription polymerase chain reaction. In primary keratinocyte, HaCaT, and A431 carcinoma cell cultures none of the 10 other growth factors or extracellular matrices studied were able to upregulate epilysin expression. Our results suggest that epilysin expression is tightly spatially and temporally regulated during wound repair. Although the in vivo substrates of epilysin are not known at present, its expression pattern suggests that it may be needed to restructure the basement membrane or to degrade adhesive proteins between keratinocytes to supply new cells for the migrating front.

Blister↗

PubMedDate not supplied

PDGF-BB induces expression of LTBP-1 but not TGF-beta1 in a rat cirrhotic fat storing cell line.

TGF-beta, a profibrogenic cytokine is predominantly secreted as a latent molecule complexed with one of the latent TGF-beta binding proteins (LTBP). Due to the proposed functions of LTBP-1 and -3 in regulating TGF-beta-bioavailability and -activity, we investigated the effects of PDGF-BB and TGF-beta1 on their expression levels in Cirrhotic fat storing cells (CFSC). CFSC basally express LTBP-1 and -3 and TGF-beta1. LTBP-1 colocalizes with LAP and the cells secrete some active TGF-beta1. Promoter studies showed no strong induction of the LTBP-1 promoters after stimulation, although mRNA and protein levels were increased by PDGF-BB treatment without affecting TGF-beta1 expression. Vice versa, TGF-beta1 treatment did not alter LTBP-1 expression while an autocrine induction was found. Our data indicate that LTBP-1 but not TGF-beta1 is induced by PDGF-BB and that TGF-beta1 autoinduction does not affect the expression of LTBP-beta1. This divergent regulation may represent an important mechanism for modulation of TGF-beta bioavailability.

Adipocytes↗