Female sex steroid hormones and pregnancy regulate receptors for calcitonin gene-related peptide in rat mesenteric arteries, but not in aorta.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator neuropeptide known to be involved in the regulation of vascular tone. Results of previous studies from our laboratory and others suggest that vascular sensitivity to CGRP is enhanced during pregnancy and that the female sex steroid hormones estradiol-17beta (E2) and progesterone (P4) may be involved in this process. We hypothesized that CGRP receptors in the mesenteric artery are increased during pregnancy and with sex steroid hormone treatments. In the present study, we investigated whether pregnancy and female sex steroid hormones modulate the CGRP-receptors CGRP-A and CGRP-B in the mesenteric artery in the rat. The CGRP-A receptor consists of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein 1 (RAMP1); however, the CGRP-B receptor needs to be further characterized. Messenger RNA levels for CRLR and RAMP1 were assessed by reverse transcription-polymerase chain reaction, and CGRP-B receptor proteins levels were determined by Western blot analysis. In addition, [125I]CGRP binding was measured by Scatchard analysis. Both mRNA for CGRP-A (CRLR and RAMP1) and the protein for CGRP-B receptors in mesenteric arteries were increased with pregnancy compared to nonpregnant, diestrous animals. A P4 antagonist, RU-486, downregulated and P4 upregulated these receptors in mesenteric arteries (P < 0.05) in pregnant rats. In adult ovariectomized rats, P4 upregulated CRLR and RAMP1 mRNA levels as well as [125I]CGRP-binding sites. The CGRP-B-receptor protein levels were significantly (P < 0.05) elevated by P4 and by combined E2 and P4 treatment. Together with earlier findings, these data suggest that increases in the expression of CGRP-A (CRLR and RAMP1) and CGRP-B receptors in mesenteric arteries may be important in reducing vascular resistance and in vascular adaptations that occur during pregnancy; in addition, P4 may be involved in this process.

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-1) or P13 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.

Heregulin regulates cytoskeletal reorganization and cell migration through the p21-activated kinase-1 via phosphatidylinositol-3 kinase.

The mechanisms through which heregulin (HRG) regulates the activities of breast cancer cells are currently unknown. We demonstrate that HRG stimulation of noninvasive breast cancer cells enhanced the conversion of globular to filamentous actin and the formation of membrane ruffles, stress fibers, filopodia, and lamellipodia and accompanied by increased cell migration. In addition, HRG triggered a rapid stimulation of p21-activated kinase1 (PAK1) activity and its redistribution into the leading edges of motile cells. The HRG-induced stimulation of PAK1 kinase activity followed phosphatidylinositol-3 kinase (PI-3 kinase) activation. Inhibition of PI-3 kinase activity blocked the activation of PAK1 kinase and also blocked cell migration in response to HRG. Furthermore, direct inhibition of PAK1 functions by the dominant-negative mutant suppressed the capacity of HRG to reorganize actin cytoskeleon structures. We also demonstrated that HRG stimulation promoted physical interactions between PAK1, actin, and human epidermal growth factor receptor 2 (HER2) receptors, and these interactions were dependent on the activation of PI-3 kinase. The blockade of HER2 receptor by an anti-HER2 monoclonal antibody resulted in the inhibition of HRG-mediated stimulation of PI-3 kinase/PAK pathway and also the formation of motile actin cytoskeleton structures but not extracellular signal-regulated kinases. These findings suggest a role of PI-3 kinase/PAK1-dependent reorganization of the cortical actin cytoskeleton in HRG-mediated increased cell migration, and these changes may have significant consequences leading to enhanced invasion by breast cancer cells.

Epidermal growth factor and amphiregulin up-regulate matrix metalloproteinase-9 (MMP-9) in human breast cancer cells.

The EGF family of proteins encompasses several polypeptides such as epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), amphiregulin (AR) and heregulin (HRG-beta 1). These polypeptides regulate proliferation in breast cancer cells through interaction with membrane receptors. It has been previously shown that high EGF receptor number correlates with aggressive behavior and increased metastasis in human breast cancer. In the present study, we investigated the association between EGF and EGF-like ligand-induced DNA synthesis and secretion of MMP-9 and MMP-2 in metastatic SKBR-3 and non-metastatic MCF-7 breast cancer cells. Exposure of SKBR-3 cells to EGF or AR induces expression of MMP-9 but has no effect on MMP-2 secretion. In contrast to EGF and AR, HRG had no effect on gelatinase induction. None of the EGF polypeptides had any effect on gelatinase induction in MCF-7 non-metastatic breast cancer cells. While a relatively specific inhibitor of EGF receptor tyrosine kinase, PD 153035, inhibited EGF-, AR- and HRG-induced cell proliferation, it had no effect on MMP-9 induced by EGF and AR. Experimental evidence suggests that signaling mechanisms for cell proliferation and MMP-9 induction are mediated by different pathways down-stream of EGF receptor autophosphorylation or that low levels of EGF-induced signal that escape inhibition are sufficient to induce MMP-9 but unable to support cell proliferation. In addition, our results suggest that EGF and AR may modulate invasion of metastatic breast cancer cells by increasing the expression of MMPs.