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Cell-cell interactions in the local control of seminiferous tubule contractility.

Since peritubular smooth muscle cells (PSMCs) have been characterized, isolated and cultured in homogeneous populations, the regulation of seminiferous epithelium contractility has received increasing attention. The present article reports and discusses experimental evidence demonstrating that: (1) PSMCs express two types of high affinity receptors for endothelin (ET-A and ET-B), both responsible for calcium-mediated cell contraction, but coupled to partially different transduction pathways; (2) the production of endothelin by seminiferous epithelium follows a finely regulated spatial and temporal pattern, which is based on the cyclic expression of endothelin-converting-enzyme in Sertoli cells; and (3) a further local factor, PDGF-BB, is capable of stimulating PSMC contraction when acutely administered and induces cell hypertrophy and potentiation of contractile phenotype when chronically administered, in the absence of any proliferative response. The study of the mechanisms through which PSMC contractile activity and differentiated state is locally controlled may be of potential relevance to our knowledge of how the efficiency of tubular output is regulated in normal and pathological conditions.

Animals↗

Fever induced by platelet-derived growth factor, in contrast to fever induced by lipopolysaccharide, depends only on nitric oxide, but not on carbon monoxide pathway.

Platelet-derived growth factor (PDGF) is a multifunctional protein which is known to induce a febrile response when injected intracerebroventricularly. The gaseous neurotransmitters, nitric oxide (NO) and carbon monoxide (CO), are both known to exert thermoregulatory effects and to participate in lipopolysaccharide-induced fever. In this study, we investigated the role of NO and CO in the febrile response to PDGF-BB in rats. Intracerebroventricular (i.c.v.) injection of PDGF-BB produced a dose-dependent increase in body temperature. This increase in body temperature induced by PDGF-BB was exacerbated by N(G)-nitro-L-arginine methyl ester (L-NAME-a nonselective NO synthase inhibitor) and S-methyl-L-thiocitrulline treatment [SMTC-a neuronal NOS (nNOS) selective inhibitor], but not by aminoguanidine treatment [an inducible NOS (iNOS) selective inhibitor]. Zinc deuteroporphyrin 2,4-bis glycol treatment (ZnDPBG-a nonselective heme oxygenase (HO) blocker) did not affect PDGF-BB fever. Our data indicate that the NO but not the CO pathway participates in PDGF-BB fever. Furthermore, our data show that nNOS is the NOS isoform responsible for NO synthesis in this response.

Animals↗

Heparinase III limits rat arterial smooth muscle cell proliferation in vitro and in vivo.

Heparinase III degrades heparan sulfate proteoglycans, which are co-receptors for growth factors that stimulate arterial proliferation. We assessed the ability of locally-delivered heparinase III to limit medial vascular smooth muscle cell proliferation induced by balloon catheter injury in rat carotid arteries. Whereas vehicle-treated arteries showed 12% of smooth muscle cells proliferating after 2 days, heparinase III (0.022-5.7 mg/kg) treated arteries showed 0.8-4%. Chemically-inactivated heparinase III did not limit proliferation. In isolated rat A10 vascular smooth muscle cells, heparinase III (1 IU/ml) inhibited both PDGF-BB and bFGF mediated increases in proliferation and migration. These results suggest that heparinase III can limit proliferation by affecting heparan sulfate proteoglycan binding growth factors following arterial injury.

Animals↗

Endothelin ET(A) receptor antagonist reverses the inhibitory effect of platelet-derived growth factor on cytokine-induced nitric oxide production.

Cytokines and cytokine-induced nitric oxide (NO) play important roles in inflammatory glomerular diseases, and both platelet-derived growth factor and transforming growth factor-beta inhibit cytokine-induced NO production. In this study, we demonstrated that a selective endothelin ET(A) receptor antagonist, BQ-485 (Hexahydro-1H-azepinylcarbonyl-Leu-D-Trp-D-Trp-OH), reversed the inhibitory effect of platelet-derived growth factor on cytokine-induced NO production, but not that of transforming growth factor-beta. Our findings suggest a difference between the inhibitory mechanisms of platelet-derived growth factor and transforming growth factor-beta on cytokine-induced NO production.

Animals↗

SKP-450 inhibits migration and DNA synthesis stimulated by oxidized low density lipoprotein in smooth muscle cells.

This study was carried out to examine the inhibitory effects of SKP-450 (2-[2"(1", 3"-dioxolone)-2-methyl]-4-(2'-oxo-1'-pyrrolidinyl)-6-nitro-2H-1-be nzo pyran), a potassium channel opener, on the proliferation and migration stimulated by oxidized low density lipoprotein (LDL) of cultured smooth muscle cells of Wistar Kyoto rat aorta. SKP-450 (10(-7) and 10(-6) M) as well as probucol (10(-7)-10(-5) M) reduced the production of thiobarbituric acid reactive substances from LDL submitted to CuSO(4) (10 microM). The increased [3H]thymidine incorporation and migration (chemotactic and wound-edge) of the cultured smooth muscle cells in association with increased production of platelet-derived growth factor (PDGF)-BB-like immunoreactivity stimulated by oxidized LDL were significantly reduced by SKP-450 (10(-7)-10(-6) M). Inhibition by SKP-450 of the oxidized LDL-stimulated [3H]thymidine incorporation was antagonized by iberiotoxin (10(-7) M), but not by glibenclamide (10(-6) M), suggestive of mediation of Ca(2+)-activated K(+) channel opening in the action of SKP-450. Taken together, SKP-450 inhibited the proliferation and migration of the smooth muscle cells as well as PDGF production stimulated by oxidized LDL, accompanying with its antiperoxidative action.

Animals↗

Cyclooxygenase-independent inhibition of smooth muscle cell mitogenesis by ibuprofen.

The aryl-propionic acid derivative, ketoprofen, has been shown to inhibit fibroblast growth by a cylooxygenase-dependent mechanism [Sánchez, T., Moreno, J.J., 1999. S(+) enantiomer inhibits prostaglandin production and cell growth in 3T6 fibroblast cultures. Eur. J. Pharmacol. 370, 63-67]. The present study demonstrates that ibuprofen, another aryl-propionic acid derivative, inhibited platelet-derived growth factor-BB (20 ng/ml)-induced mitogenesis of cultured bovine coronary artery smooth muscle cells in a stereo-independent manner. In addition, pretreatment of the cells with indomethacin (3 microM) did not affect the inhibitory effects of ibuprofen enantiomers on smooth muscle cell mitogenesis. Thus, aryl-propionic acid-type cyclooxygenase inhibitors can inhibit cell proliferation by both, cyclooxygenase-dependent and -independent ways.

6-Ketoprostaglandin F1 alpha↗

Expression of minichromosome maintenance proteins in vascular smooth muscle cells is ERK/MAPK dependent.

Proliferation of vascular smooth muscle cells (VSMC) represents a key event for the pathogenesis of postangioplasty restenosis. Minichromosome maintenance proteins (MCM) form essential components of the prereplicative complex at DNA replication origins and are regulated by E2F. The present studies were designed to investigate the signal transduction pathways controlling the expression of MCM6 and MCM7 in VSMC in response to mitogenic stimuli. MCM6 and MCM7 expression was substantially increased after stimulation with platelet-derived growth factor-BB and insulin. Pretreatment with PD98059, a specific inhibitor of the extracellular signal-regulated kinases (ERK)-mitogen-activated protein kinase (MAPK), competely inhibited the mitogen-induced MCM6 and MCM7 mRNA and protein expression, demonstrating a critical role for this pathway in transmitting transmembrane signals required for the initiation of DNA replication. The p38MAPK inhibitor SB203580, the phosphatidylinositol 3 kinase (PI3-kinase) pathway inhibitor wortmannin, and the protein kinase C pathway (PKC) inhibitor Gö 6976 did not significantly affect mitogen-induced MCM6 and MCM7 expression. Transient transfection experiments revealed that PD98059 inhibited mitogen-induced MCM6 and MCM7 transcriptional activation. In addition, blockade of ERK/MAPK signaling with PD98059 strongly inhibited phosphorylation of the retinoblastoma protein (Rb) and activity of a luciferase reporter plasmid driven by multiple E2F elements. Inhibition of mitogen-induced MCM6 and MCM7 expression by PD98059 was reversed by ectopic overexpression of E2F, indicating that ERK/MAPK signaling is required for events that occur upstream of E2F release from phosphorylated Rb. In combination, these data demonstrate that the ERK/MAPK signal transduction pathway plays a central role in regulating E2F-dependent MCM expression and DNA replication in VSMC.

Becaplermin↗

PDGF-induced Akt phosphorylation does not activate NF-kappa B in human vascular smooth muscle cells and fibroblasts.

A recent report suggested that platelet-derived growth factor (PDGF) activates nuclear factor-kappa B (NF-kappa B) by phosphorylation of the protein kinase Akt [Romashkova and Makarov, Nature 401 (1999) 86-90]. The present study investigates the role of Akt in the activation of NF-kappa B by tumor necrosis factor-alpha (TNF alpha, 10 ng/ml) and PDGF-BB (20 ng/ml) in human vascular smooth muscle cells (SMC), skin and foreskin fibroblasts. TNF alpha stimulated serine phosphorylation and degradation of the inhibitory protein I kappa B alpha and strongly induced nuclear NF-kappa B translocation and binding activity. PDGF did not induce serine phosphorylation or degradation of I kappa B alpha and did not enhance binding activity of NF-kappa B. In contrast, stimulation with PDGF resulted in a marked phosphorylation of Akt, but no Akt phosphorylation occurred after stimulation with TNF alpha. These data suggest that Akt phosphorylation is not involved in NF-kappa B activation in human SMC and fibroblasts.

Becaplermin↗

PDGF induces osteoprotegerin expression in vascular smooth muscle cells by multiple signal pathways.

Osteoprotegerin (OPG) is a key regulator of osteoclastogenesis. Recent reports suggest that OPG may function as a protector of arterial calcification and survival of endothelial cells. However, the role and expression of OPG in vascular wall is unclear. Here we report that OPG was highly expressed in vascular smooth muscle cells (VSMC) but not in endothelial cells. Platelet-derived growth factor (PDGF), basic fibroblast growth factor, angiotensin II, tumor necrosis factor alpha and interleukin-1beta upregulated OPG expression in VSMC. Moreover, inhibition of phosphatidylinositol 3-kinase/Akt or P38-signal pathway abrogated PDGF-induced OPG expression. Our results suggest that OPG may be an important determinant of vascular homeostasis.

Angiotensin II↗

Platelet-derived growth factor induces p21/WAF1 promoter in vascular smooth muscle cells via activation of an Sp1 site.

Many studies suggested that cyclin-dependent kinase inhibitor (CDKI) p21 acts as a universal inhibitor of cyclin/CDK catalytic activity. This protein has also been shown to be a component of active cyclin/CDK complexes. In addition, it has recently been suggested that p21 serves as an assembly factor in platelet-derived growth factor (PDGF)-stimulated vascular smooth muscle cells (VSMC). However, little is known concerning the molecular mechanisms by which PDGF induces p21 gene expression in VSMC. In this report we demonstrate that PDGF induces the p21 expression at both the mRNA and protein levels. This increase in p21 gene expression was due to activation of the p21 promoter by PDGF. Through both deletion and mutation analysis of the p21 promoter, we defined a 10-bp sequence that is required for the activation of the p21 promoter by PDGF. In addition, gel shift and supershift assays demonstrated that this PDGF-responsive element binds specifically to the transcription factor Sp1. These results demonstrate that Sp1 mediates PDGF-induced p21 gene expression in VSMC. Moreover, immunoblot and immunoprecipitation analysis showed that the level of hyperphosphorylated retinoblastoma protein (Rb) is increased and the protein is physically associated with Sp1 in PDGF-treated cells, indicating that phosphorylated Rb may play a role in regulating Sp1 to activate p21 expression.

Base Sequence↗

Synergistic effects of transforming growth factor-beta on the expression of c-fms, macrophage colony-stimulating factor receptor gene, in vascular smooth muscle cells.

Vascular smooth muscle cells (SMC) transform to foam cells in the process of atherosclerosis. We have reported that SMC derived from the intima of atherosclerotic lesions express c-fms, macrophage colony-stimulating factor receptor gene, which is not normally expressed in medial SMC. In the present study, we demonstrated that transforming growth factor-beta (TGF-beta) synergistically induced expression of c-fms in the presence of platelet-derived growth factor-BB in human medial SMC, a level comparable to that observed in the intima. The induction of c-fms was not inhibited by protein kinase C (PKC) inhibitor, suggesting that TGF-beta induces c-fms via a PKC-independent pathway. These results suggest that TGF-beta plays an important role in the phenotypic change of smooth muscle cells to macrophage-like cells in the process of atherosclerosis.

Becaplermin↗

Aggregation of PDGF-beta receptors in human skin fibroblasts: characterization by image correlation spectroscopy (ICS).

Receptor aggregation is believed to be an important, early step when growth factors such as PDGF stimulate proliferation and differentiation of cell populations. To investigate receptor aggregation, we utilized a novel biophysical technique, image correlation spectroscopy, to study the distribution and aggregation state of PDGF-beta receptors on the surface of human dermal fibroblasts under various experimental conditions. It was found that the cell surface receptors were pre-clustered at 4 degrees C and receptor aggregation increased for samples measured at 37 degrees C. Treatment with PDGF-BB had no measurable effect on the receptor aggregation state. The results also indicate that additions of 10% serum or an inhibitor of tyrosine kinase activity, may disperse the receptors. The results of this study are consistent with organization of PDGF-beta receptors in pre-existing membrane domains.

Becaplermin↗

Urokinase plasminogen activator induces smooth muscle cell migration: key role of growth factor-like domain.

We defined the role of urokinase plasminogen activator (uPA) and its growth factor-like domain (GFD) in stimulating smooth muscle cell (SMC) migration. Recombinant uPA (r-uPA) stimulated migration approximately 3-fold whilst the recombinant uPA mutant containing an altered GFD (r-uPAmut) was ineffective. Both uPA variants bound to the same high affinity receptor in a competitive manner. FGF-2- and PDGF-BB-induced migration was also dependent on uPA, their effects being antagonized by addition of a uPA-neutralizing antibody or the r-uPAmut. Thus r-uPA is chemotactic for SMC and stimulation of cell migration by PDGF-BB and FGF-2 is dependent on uPA. The GFD of uPA is essential for its chemotactic effects.

Amino Acid Sequence↗

A cyclic peptide analogue of loop III of PDGF-BB causes apoptosis in human fibroblasts.

A cyclic peptide analogue of platelet-derived growth factor-BB (PDGF-BB), P1 [77IVRKK81-C-73RKIE76], has recently been shown to inhibit specifically [125I]PDGF-BB/receptor binding, and PDGF-BB-induced DNA synthesis in cells expressing PDGF receptors. Here we demonstrate that P1 induces apoptosis in exponentially growing human fibroblasts as confirmed by characteristic changes in cell and nuclear morphology, by TUNEL staining and by flow cytometry. Following incubation with P1 (100 microM), the percentage of cells exhibiting DNA fragmentation increased from 24% after 8 h to 76% after 28 h as exponentially growing cells progressed through the cell cycle. We conclude from these findings taken together that apoptosis accounts for the major proportion of P1-induced cell death. Omission of the Cys residue from P1 or replacement by Ser did not alter the potency of the peptide confirming that peptide dimerisation is not important for its activity. PDGF-BB, EGF, FGF, thrombin and foetal bovine serum were not able to rescue cells from the effects of P1. P1 is a useful tool for investigation of the balance of cellular proliferation/apoptosis in wound healing, atherosclerosis and restenosis, and constitutes a basis from which to design compounds with greater potency.

Animals↗

Vascular types I and II transforming growth factor-beta receptor expression: differential dependency on tyrosine kinases during induction by TGF-beta.

Recent evidence indicates that the type II transforming growth factor-beta (TGF-beta) receptor (TbetaRII) is a serine-threonine-tyrosine kinase. However, the significance of its tyrosine kinase is unclear. We investigated in vascular smooth muscle cells the effects of tyrosine kinase inhibition on the expression of TGF-beta receptor types I (ALK-5) and II (TbetaRII) mRNA, induced by TGF-beta1. TGF-beta1 elevated ALK-5 mRNA levels 5-fold; essentially similar TGF-beta1-dependent elevations were observed with growth factors, PDGF-BB and FGF-2. The tyrosine kinase inhibitor genistein abolished these TGF-beta1 and growth factor responses. TGF-beta1 also elevated TbetaRII mRNA levels which were not inhibited by genistein. We conclude that tyrosine kinases participate in defining how cells respond to TGF-beta.

Activin Receptors, Type I↗

Rapid activation of the novel serine/threonine protein kinase, protein kinase D by phorbol esters, angiotensin II and PDGF-BB in vascular smooth muscle cells.

Protein kinase D (PKD) is a novel serine/threonine kinase structurally distinct from all protein kinase C (PKC) isoforms but which like classic and novel PKCs is activated by phorbol esters and diacylglycerol. This study investigated the regulation of PKD in vascular smooth muscle cells (VSMC) by physiological regulators of VSMC function and growth factors. Treatment of rabbit aortic VSMC with phorbol ester, angiotensin II and PDGF-BB all stimulated PKD activity in a time- and concentration-dependent manner in VSMC. The effect of angiotensin II was particularly rapid and potent (maximum stimulation within 1 min and at 0.5 nM). In contrast, the maximum effect of PDGF-BB was obtained after 5 min. Other factors, including basic FGF, IGF-I, IGF-II, endothelin-1 and endothelin-2, had no effect on PKD activity in VSMC. These results show for the first time that PKD activity is regulated in VSMC, and is activated by the vasoconstrictor angiotensin II. PKD may be an important mediator for the biological function(s) of one or more PKC isoforms in VSMC and/or may represent a component of a novel PKC-independent signalling pathway in VSMC.

Angiotensin II↗

Low molecular weight phosphotyrosine protein phosphatase translocation during cell stimulation with platelet-derived growth factor.

Low Mr phosphotyrosine protein phosphatase (PTP) is a cytosolic enzyme whose activity upon platelet-derived growth factor (PDGF) and insulin receptors has been demonstrated in vivo. In our study we demonstrate that this enzyme, both naturally expressed and overexpressed in NIH/3T3 fibroblasts, translocates from the cytosol to the Triton X-100 insoluble fraction following stimulation with PDGF. It emerges that the phosphorylation of a defined population of PDGF receptors, which is localized in this fraction and seems to be endowed with peculiar features and functions, is particularly affected by low Mr PTP overexpression.

3T3 Cells↗

Raf-1 is activated by the p38 mitogen-activated protein kinase inhibitor, SB203580.

SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imi dazole) is widely used as a specific inhibitor of p38 mitogen-activated protein kinase (MAPK). Here, we report that SB203580 activates the serine/threonine kinase Raf-1 in quiescent smooth muscle cells in a dose-dependent fashion. The concentrations of SB203580 required lie above those necessary to inhibit p38 MAPK and we were unable to detect basal levels of active p38 MAPK. SB203580 does not directly activate Raf-1 in vitro, and fails to activate Ras, MEK, and ERK in intact cells. In vitro, however, SB203580-stimulated Raf-1 activates MEK1 in a coupled assay. We conclude that activation of Raf-1 by SB203580 is not mediated by an inhibition of p38 MAPK, is Ras-independent, and is uncoupled from MEK/ERK signaling.

3T3 Cells↗