Clement A. Diglio

Mitogen-activated protein kinase (MAPK) regulates the expression of progelatinase B (MMP-9) in breast epithelial cells

Mitogen‐activated protein kinases (MAPKs) play a major role in the mitogenic signal transduction pathway and are essential components of both growth and differentiation. Constitutive activation of the MAPK cascade is associated with the carcinogenesis and metastasis of human breast and renal cell carcinomas. The gelatinases B (MMP‐9) and A (MMP‐2) are 2 members of the matrix metalloproteinase (MMPs) family which are expressed in human cancers and thought to play a critical role in tumor cell invasion and metastasis. In a previous study, we have shown that EGF and amphiregulin upregulate MMP‐9 in metastatic SKBR‐3 cells but have no effect on MMP‐2 secretion. We now investigated specific step(s) in EGF‐induced signalling associated with regulation of cell proliferation and MMP‐9 induction. EGF‐induced signalling in SKBR‐3 cells was blocked by relatively specific inhibitors either on ras (FPT inhibitor‐I) or PI3 kinase (Wortmannin) or by reduction in EGF‐induced tyrosine kinase activity (RG 13022). Blocking these signalling pathways significantly inhibited of EGF‐induced cell proliferation but only partially reduced in EGF‐induced MMP‐9 secretion. In contrast, when SKBR‐3 cells were exposed to MEK inhibitor (PD 98059) or MAPK inhibitors (Apigenin or MAPK antisense phosphorothioate oligodeoxynucleotides), EGF‐induced cell proliferation, MMP‐9 induction and invasion through reconstituted basement membrane were significantly reduced. Our results suggest that interfering with MAPK activity may provide a novel means of controlling growth and invasiveness of tumors in which the signalling cascade is activated. Int. J. Cancer 82:268–273, 1999.

Regulation of endothelin-1 mRNA by angiotensin II in rat heart endothelial cells

In a series of experiments carried out in cultured endothelial cells derived from rat hearts (RHE), angiotensin II (AII) is shown to stimulate preproendothelin-1 mRNA in a dose- and time-dependent manner. The induction of preproendothelin-1 mRNA is rapid, reaching a maximal level 1 h after the addition of AII (1 x 10(-8) M). The mRNA levels decline rapidly to basal levels in 4 h. The addition of Losartan (Dup 753; 1 x 10(-6) M), an AII receptor (type I) antagonist, blocks the AII effect. Calphostin C, a potent protein kinase C inhibitor, is able to abolish this effect of AII suggesting that the induction of preproendothelin-1 mRNA is mediated by a protein kinase C-dependent pathway. Since endothelial cells line the inner surface of the myocardium and blood vessels and sense the rise of AII associated with renovascular hypertension at the endothelial surface, these data suggest that endothelin which is produced by RHE cells in response to AII could be an important mediator which may play a role in modulating gene expression in AII-mediated cardiac hypertrophy.

Angiotensin II induces TIMP-1 production in rat heart endothelial cells.

Angiotensin II (AII) was found to upregulate tissue inhibitor of metalloproteineses-1 (TIMP-1) gene expression in rat heart endothelial cells in a dose and time-dependent manner. The maximal stimulation of TIMP-1 mRNA was achieved by 2 h after the addition of AII. This effect was blocked by losartan, an AT1 receptor antagonist and by calphostin C, a protein kinase C inhibitor. Addition of cycloheximide superinduced and actinomycin D abolished the induction. These results suggest that AII stimulates TIMP-1 production by a protein kinase C dependent pathway which is dependent upon de novo RNA synthesis. Immunoprecipitation experiment showed an enhanced band of 28 kDa from the conditioned medium of AII-treated cultures. Immunoblot analysis revealed that TIMP-1 was detectable in the conditioned medium 4 h after AII stimulation. Since endothelial cells line the blood vessels and sense the rise in AII associated with hypertension, the TIMP-1 released by these cells may provide an initial trigger leading to cardiac fibrosis in angiotensin-renin dependent hypertension.

Effect of Growth Factors on Collagen Metabolism in Cultured Human Heart Fibroblasts

Using human heart fibroblasts (HHF), we studied the effect of basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) on the gene expression of type I collagen, collagenase and tissue inhibitor of metalloproteinases (TIMP). Initially, treatment of HHF with bFGF alone (10 ng/ml) resulted in elevated secretion of collagenase into the culture medium. Subsequent treatment of HHF with TGF-beta in combination with bFGF suppressed collagenase secretion. Northern blot analysis reinforced this observation by revealing an enhancement of the steady-state mRNA level of collagenase in response to bFGF. In order to examine if the collagenase gene was affected by bFGF at the transcriptional level, transfection experiments were carried out with a plasmid containing collagenase promoter linked to chloramphenicol acetyltransferase gene (CAT). Basic FGF stimulated CAT activity by four-fold, indicating increased promoter activity whereas the combination of TGF-beta and bFGF resulted in decreased CAT activity. TGF-beta was shown to increase type I collagen and TIMP mRNA levels by 2.5- and 2.1-fold, respectively. These results suggest that TGF-beta and bFGF may play a pivotal role in regulating collagen metabolism in HHF.

Thrombin enhances tumor cell adhesive and metastatic properties via increased αIIbβ3 expression on the cell surface

Abstract The association between blood coagulation and cancer growth and metastatic dissemination is not yet completely understood. In this study we demonstrate that thrombin is capable of enhancing tumor cell adhesive properties and thereby increases tumor cell metastatic potential. Following exposure to α-thrombin, Walker 256 carcinosarcoma cells and B16 amelanotic melanoma cells became more adherent to both endothelial cell monolayers and the subendothelial matrix component, fibronectin. Preincubation of W256 and B16a cells with doses of α-thrombin from 0.01 to 10.0 U/ml produced a bell shape dose-response curve with the maximal effect (a 2–5-fold increase in adhesion) observed at 0.1 U/ml (corresponding to 0.8 nM). Complexes of α-thrombin with its inhibitors, hirudin and antithrombin III-heparin, diminished its effect on tumor cell adhesion. The effect of thrombin on tumor cell adhesion may be mediated by the αIIbβ3 integrin as thrombin increased cell surface expression of the αIIbβ3 complex. The significance of the in vitro observations was further substantiated by results of in vivo studies. Pretreatment of B16a cells with α-thrombin resulted in a 2-fold increase in the number of metastatic lung colonies in an experimental metastasis model. The data indicate a new role for thrombin in the metastatic spread of cancer.

Identification of muscarinic receptors in rat cerebral cortical microvessels.

Abstract: Microvessels isolated from rat cerebral cortex consist mainly of capillaries (>85%). Fresh, intact microvessel preparations have been analyzed by radioligand binding techniques for muscarinic receptors. Scatchard analysis of specific quinuclidinyl benzilate (QNB) binding indicates that microvessels possess a large number of muscarinic sites (914 fmol/mg protein) of high affinity (Kd= 0.034 nM). The association and dissociation rate constants (0.37 min−1 nM−1 and 0.0067 min−1, respectively) yield an equilibrium Kd of 0.018 nM. Displacement of [3H]QNB by muscarinic ligands and control substances is typical of muscarinic receptors. The results indicate that cerebral microvessels possess a large population of muscarinic receptors.

ISolation and characterization of cerebral resistance vessel endothelium in culture

Organ-derived endothelia have been shown to exhibit distinct patterns of morphology and growth responsiveness in vitro. This report describes the development, cloning and establishment of long-term serial cultures of rat vascular endothelial cells derived from cerebrocortical resistance vessels (small arteries and arterioles). Modification of our previous published technique for establishing resistance vessel-derived smooth muscle cells (RV-SMC) resulted in enhanced levels of endothelial outgrowth from collagenase-treated microvessel fragments. Although primary culture growth consisted predominantly of SMC, subsequent subcultivation of these cultures revealed the presence of distinct endothelial cell clusters within the SMC monolayer. Serial cloning of these isolates resulted in a homogeneous population of cells with the characteristic endothelial cobblestone growth pattern and positive immunofluorescence for factor VIII-related antigen. Previously established RV-SMC frozen stocks provided an additional source for obtaining resistance vessel endothelial cells. This was made possible by the slow proliferation rate of early-passage RV-SMC and their inability to withstand freezing procedures. Endothelial cells from both preparations were identical and designated resistance vessel derived endothelial cells RV-EC. Upon long-term cultivation (> P15), confluent RV-EC cultures expressed spontaneous multicellular cord development that stained positive for factor VIII-related antigen. Cell growth studies demonstrated that RV-EC were capable of significant growth when maintained in serum-free conditions. Growth kinetics using serum-free conditioned medium demonstrated mitogenic activity indicating the presence of an autocrine growth factor. Increase growth responsiveness was also noted in RV-EC when treated with a variety of peptide growth factors. These results indicate that resistance vessel endothelium can be successfully isolated and maintained in long-term serial cultures. Furthermore, the availability of cultured EC and SMC from this unique microvascular site will enable examination of cerebrovascular endothelial-smooth muscle cell interactions in vitro and may help to elucidate the mechanisms of altered vascular function in disease states.

Fatty acid modulation of tumor cell adhesion to microvessel endothelium and experimental metastasis

Tumor cell interaction with the endothelium of the vessel wall is a rate limiting step in metastasis. The fatty acid modulation of this interaction was investigated in low (LM) and high (HM) metastatic B16 amelanotic melanoma (B16a) cells. 12(S)-HETE increased the adhesion of LM cells to endothelium derived from pulmonary microvessels. All other monohydroxy and dihydroxy fatty acids were ineffective. LTB4 induced a modest stimulation but LTC4, LTD4, LTE4 as well as LXA4 and LXB4 were ineffective. The 12(S)-HETE enhanced adhesion of B16a cells was inhibited by pretreatment with 13(S)-HODE but not by 13(R)-, 9(S)-HODE or 13-OXO-ODE. 13(S)-HODE decreased adhesion of HM B16a cells to endothelium. 12(S)-HETE enhanced surface expression of integrin alpha IIb beta 3 and monoclonal antibodies against this integrin but not against alpha 5 beta 1, blocked enhanced but not basal adhesion to endothelium. Intravenous injection of 12(S)-HETE treated LM cells resulted in increased lung colonization (experimental metastasis). This effect was specific for 12(S)-HETE and was inhibited by 13(S)-HODE but not by other HODEs. 12(S)-HETE also enhanced lung colonization by HM cells and 13(S)-HODE decreased lung colonization by HM cells. Our results suggest a highly specific bidirectional modulation of metastatic phenotype and lung colonization by 12(S)-HETE and 13(S)-HODE.

αIIbβ3 Integrin expression and function in subpopulations of murine tumors

Abstract Subpopulations of B16 amelanotic melanoma (B16a) cells, isolated by centrifugal elutriation from enzymatically dispersed solid tumors, demonstrated different abilities to form lung colonies when injected intravenously. In contrast, no differences in experimental metastasis were observed among subpopulations obtained from Lewis lung (3LL) tumors. Lung colonization by B16a and 3LL subpopulations correlated positively with observed differences (B16a) or lack of differences (3LL) in tumor cell ability to induce aggregation of homologous platelets, to adhere to subendothelial matrix or fibronectin, and with the percentage of cells in the G 2 M phase of the cell cycle. Both B16a and 3LL cells express αIIbβ3 integrin receptors; however, differences in the receptor expression level were found only among B16a subpopulations. Comparison of the amount of αIIbβ3 receptor expressed on cell surface with tumor cell ability to induce platelet aggregation (TCIPA) and to adhere to fibronectin or subendothelial matrix revealed a positive correlation. Pretreatment of tumor cells with αIIbβ3 -specific antibodies inhibited tumor cell matrix adhesion, TCIPA, and lung colony formation. We propose that αIIbβ3 integrin receptor expression, tumor cell matrix adhesion, and tumor cell-induced platelet aggregation can be important parameters to indicate the metastatic potential of some tumor cells and that the αIIbβ3 is a multifunctional receptor involved in both tumor cell-matrix and tumor cell-platelet interactions. Further, the correlation among cell cycle phase, metastatic ability, and receptor expression suggests that metastatic propensity may be transiently expressed and/or increased in some tumor cell subpopulations.

12(S)-HETE-induced microvascular endothelial cell retraction results from PKC-dependent rearrangement of cytoskeletal elements and αVβ3 integrins

Abstract 12(S)-HETE, a lipoxygenase metabolite of arachidonic acid, has been demonstrated to induce a reversible retraction of vascular endothelial cells (EC). 12(S)-HETE-induced microvascular EC retraction was blocked by a selective protein kinase C inhibitor, calphostin C, but not by the protein kinase A inhibitor, H8. EC exposed to 12(S)-HETE demonstrated a gradual dissolution of actin microfilaments and a decrease of vinculin-containing focal adhesions. The intermediate filaments, vimentin, also underwent extensive reorganization (i.e., filament bundling and enrichment to the cell filapodia) following 12(S)-HETE treatment. In vivo phosphorylation studies revealed that 12(S)-HETE induced a hyperphosphorylation of several major cytoskeletal proteins including myosin light chain, actin, and vimentin. The increased phosphorylation of these cytoskeletal proteins following 12(S)-HETE stimulation was abolished by calphostin C but not by H8. Confluent EC express αvβ3 in focal adhesions at both the cell body and the cell-cell borders. 12(S)-HETE induced a sequential rearrangement of the αvβ3-containing focal adhesions, resulting in a general decrease in αvβ3 integrin receptors, especially in those retracted EC. 12(S)-HETE-induced rearrangement of αvβ3 was inhibited by calphostin C but not by H8. In contrast to αvβ3, confluent EC enrich α5β1 integrin receptors primarily at the cell-cell borders, colocalizing with extracellular fibronectin and cell cortical microfilaments. 12(S)-HETE treatment also disrupted the cell-border distribution pattern of α5β1 as EC retracted, but no distinct alterations (such as time-related redistribution and quantitative differences) in α5β1 were observed.