Increase of C-Type Natriuretic Peptide Expression by Serum and Platelet-Derived Growth Factor-BB in Human Aortic Smooth Muscle Cells Is Dependent on Protein Kinase C Activation

Abstract

C-type natriuretic peptide (CNP) is produced by the vascular smooth muscle cells (SMCs) of injured and atherosclerotic arteries, in which it may exert autocrine control over SMCs by binding to its principal receptors, NPR-B and NPR-C, but few studies have examined the factors that regulate CNP expression in human SMCs. In the present report, we show that serum induces significant increases in both CNP and NPR-C transcript levels in human, but not rat SMCs in culture, and that pretreatment with either the general tyrosine kinase inhibitor genistein, the platelet-derived growth factor (PDGF) tyrosine kinase inhibitor AG 1296, or the protein kinase C (PKC) inhibitor GF109203X blocks most of the serum-induced increase in CNP. PDGF-BB also induced significant dose-dependent increases in CNP transcript that correlated temporally with the serum effect on CNP mRNA. Inhibition of several PDGF-BB signaling pathways downstream of receptor activation showed that PKC inhibition with GF109203X was almost as effective as genistein in abolishing the PDGF-BB-induced up-regulation of CNP mRNA. Furthermore, PKC activation by phorbol 12-myristate 13-acetate (PMA) produced an extremely high level of CNP mRNA that was abolished by GF109203X. Immunoreactive CNP was markedly increased in SMCs receiving 10% serum, 20 ng/ml PDGF-BB, or PMA, and was decreased in PDGF-treated and PMA-treated cells by AG 1296 and GF109203X, respectively. This report suggests that in humans, PDGF and other factors signaling through receptor tyrosine kinases and downstream activation of PKC could represent an important control for CNP expression in vascular smooth muscle.

Transforming growth factor-β1regulation of C-type natriuretic peptide expression in human vascular smooth muscle cells: dependence on TSC22D1

“…These observations suggest that in circumstances of vascular damage, CNP is secreted by SMC and that its expression is controlled by factors produced by injured endothelium, platelets, VSM, and inflammatory cells to regulate SMC function in an autocrine or paracrine fashion. We have reported the effects of several of these mediators, including PDGF and bioactive lipids and their transduction pathways on CNP expression in human SMC (11,12).…”

“…AoSMCs, which received either control or TSC22D1 siRNA as described below, were seeded onto 12-mm round glass coverslips in 24-well plates and grown in SmGM-2 before switching to serum-free SmBM for 48 h. Cells then received fresh SmBM for 24 h before immunofluorescence staining. Cells were washed with PBS, fixed in cold methanol for 20 min, and incubated with either rabbit polyclonal anti-CNP (Bachem) diluted 1:200 as previously described (11) or with rabbit polyclonal antibody against human TSC22D1 (ProteinTech Group, Chicago, IL) diluted 1:200 followed by Alexa 488-conjugated donkey anti-rabbit antibody (Invitrogen, Camarillo, CA) diluted 1:1,500 in PBS. Cells fixed on coverslips were then mounted in Vectashield (Vector Laboratories, Burlingame, CA) and photographed using a fluorescence microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan).…”

Regulation of C-type natriuretic peptide expression

“…In rat-derived AoSMC however, CNP mRNA levels were reduced to only 7% of their control value in the presence of PDGF-BB [51]. Similarly, basic fibroblast growth factor (bFGF) was shown to suppress CNP transcript levels in cultured AoSMC derived from the rat [97], but it elevated CNP transcript levels in cultured smooth muscle cells derived from the human aorta [51]. On the other hand, CNP transcript levels responded positively to basic fibroblast bFGF in smooth muscle cells derived from human aorta, but showed no response to bFGF in human coronary-artery derived smooth muscle cells [52].…”

“…Some of these factors have exhibited remarkable variability in their effects on cardiovascular CNP expression in different species and tissue types. For example, in cultured human aortic smooth muscle cells (AoSMC), CNP mRNA levels were increased as much as 50-fold by platelet-derived growth factor BB (PDGF-BB) [51]. In rat-derived AoSMC however, CNP mRNA levels were reduced to only 7% of their control value in the presence of PDGF-BB [51].…”

“…For example, in cultured human aortic smooth muscle cells (AoSMC), CNP mRNA levels were increased as much as 50-fold by platelet-derived growth factor BB (PDGF-BB) [51]. In rat-derived AoSMC however, CNP mRNA levels were reduced to only 7% of their control value in the presence of PDGF-BB [51]. Similarly, basic fibroblast growth factor (bFGF) was shown to suppress CNP transcript levels in cultured AoSMC derived from the rat [97], but it elevated CNP transcript levels in cultured smooth muscle cells derived from the human aorta [51].…”