A proteomic approach to the investigation of early events involved in the activation of vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) are mature cells that maintain great plasticity. This distinctive quality is the basis of the migration and proliferation of VSMC in cardiovascular diseases. We have investigated, via a proteomic approach, the molecular changes that promote VSMC switching from a quiescent to an activated-proliferating phenotype. In particular, we focus on the modulation in tyrosine phosphorylation that occurs in cell activation by serum or by single growth factors, such as insulin-like growth factor 1 (IGF-1) or platelet-derived growth factor (PDGF-BB). A comparison of profiles from two-dimensional polyacrylamide gel electrophoresis analysis of quiescent and activated-proliferating VSMC has revealed a number of differences in protein expression. Several differentially expressed proteins have been identified by mass spectrometry, and their changes during the time course of tyrosine phosphorylation have been documented from time zero up to 48 h after stimulus. The tyrosine-phosphorylation level generally decreases within a few minutes of stimulation, followed by a rapid dramatic recovery of some chaperones and redox enzymes, but no significant recovery for glucose metabolism enzymes. With respect to cytoskeleton components, no remarkable fluctuations have been detected at the earliest time points, except for those relating to α-actin, which displays an impressive decrease. A comparison of the early stages of cell stimulation after serum or after single growth factor administration has revealed important differences in the phosphorylation of chaperones, thereby suggesting their crucial role in VSMC activation. This work was partially supported by two FIRB 2001 project grants to Dr. G. Rainaldi and to Prof. G. Camici.     

The regulation of caveolin expression and localization by serum and heparin in vascular smooth muscle cells

Caveolae have been implicated in growth factor receptor and G-protein coupled receptor signaling in vascular cells. It has been postulated that caveolin, the structural protein of caveolae, may act as a general tyrosine kinase inhibitor by binding and inhibiting signaling molecules involved in the activation of the MAP kinase proliferation cascade. Using an in vitro model of VSMC proliferation, we found that serum stimulation caused a dose dependent decrease in both caveolin-1 and caveolin-2 protein levels in human coronary artery smooth muscle cells. Heparin, an inhibitor of VSMC proliferation, inhibited the serum-induced loss of caveolin-1 and caveolin-2. In addition, heparin caused an increase in both caveolin-1 and caveolin-2 localization to caveolae-enriched sucrose gradient membrane fractions when compared to serum alone. Taken together, caveolin may play an important role in the regulation of VSMC proliferation and heparin and serum have opposing effects on caveolin expression and localization in VSMC.     

Modulation of urokinase plasminogen activator gene expression during the transition from quiescent to proliferative state in normal mouse cells.

We have investigated the regulation of urokinase (u-PA) mRNA in quiescent mouse fibroblasts and keratinocytes stimulated to divide by the addition of serum or epidermal growth factor (EGF), respectively. Serum stimulation of quiescent fibroblasts (BALB/c 3T3 or Swiss 3T3) results in an early and transient increase of u-PA mRNA level, which precedes by several hours the onset of DNA synthesis. A similar response is elicited by EGF stimulation of quiescent keratinocytes. The increase of u-PA mRNA parallels that of c-myc mRNA, does not require protein synthesis and is at least in part due to increase in template activity of the u-PA gene. Induction of terminal differentiation of mouse keratinocytes results in a decrease of u-PA mRNA which parallels the decrease of thymidine incorporation. In conclusion, variation in the level of u-PA mRNA is seen during G0/G1 transition and correlates with the proliferative state of these normal mouse cells.     

Enhanced growth-dependent expression of TGF beta 1 and hsp70 genes in aortic smooth muscle cells from spontaneously hypertensive rats.

Enhanced proliferation of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) as compared with Wistar-Kyoto rats (WKY) persists in long-term culture and is characterized by an accelerated entry of these cells into the synthetic S phase of the cell cycle and a higher specific growth rate, particularly evident at high cell density. In the present study, we investigated by Northern blot experiments the expression of genes putatively involved in the regulation of VSMC growth. One of them is the transforming growth factor beta 1 gene (TGF beta 1), a bifunctional modulator of cell growth whose action is dependent on cell density. The accumulation of TGF beta 1 mRNA was enhanced in growing SHR cells at every density studied as early as 24 h after inoculation with a further increase at later times. Protooncogenes c-fos and c-myc, which have been implicated in G1/S phase transition, have also been investigated in VSMC by Northern blot analysis. At low cell density, calf serum stimulated c-fos and c-myc mRNA expression was comparable in WKY and SHR cells whereas at high cell density, c-fos induction was higher in VSMC from SHR. SHR VSMC respond more to mitogenic stimulation and to environmental (e.g., heat) stress, particularly when growing near saturation density. hsp70 constitutes a gene family responsive to environmental stimuli (heat) and to mitogenic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the growth factor requirements for stimulation of WI-38 cells after extended periods of density-dependent growth arrest

When cultures of WI-38 human diploid fibroblasts reach high cell densities, they cease to proliferate and enter a viable state of quiescence. WI-38 cells can remain in this quiescent state for long periods of time; however, the longer the cells remain growth arrested, the more time they require to leave G0, progress through G1, and enter S after stimulation with fresh serum. The experiments presented here compare the response of long-term quiescent WI-38 cells (stimulated 26 days after plating) and short-term quiescent WI-38 cells (stimulated 12 days after plating) to treatment with a variety of individual purified growth factors instead of whole serum. Our results show that the qualitative and quantitative growth factor requirements necessary to stimulate G1 progression and entry into S were the same for both short- and long-term quiescent WI-38 cells, in that the same defined medium (supplemented with epidermal growth factor [EGF], recombinant human insulin-like growth factor 1 [IGF-1], and dexamethasone [DEX]) stimulated both populations of cells to proliferate with the same kinetics and to the same extent as serum. However, the long-term quiescent WI-38 cells were found to exhibit a difference in the time during which either serum or these individual growth factors were required to be present during the prereplicative period. We believe that this difference may be the cause of the prolongation of the prereplicative phase after stimulation of long-term density-arrested WI-38 cells.     

Studies on the nature of protease-induced growth stimulation in normal and transformed BHK cells.

In the presence of growth-limiting serum concentrations trypsin displays mitogenic activity on actively-growing but not quiescent BHK cells. These results suggest that BHK cells arrested in G1 (G0) are not sensitive to protease-induced growth stimulation. Previous work strongly suggested that the trypsin active-site is not directly involved in its mitogenic activity on BHK cells. Additional studies on denatured trypsin fragments further indicate that the molecular conformation and size of native trypsin may not be absolutely required for mitogenic activity. Cellular multiplication induced by the addition of fresh serum to quiescent BHK cultures is not inhibited by high concentrations of soybean trypsin inhibitor. Similar to our previous findings with trypsin, it has been further observed that plasmin is not sufficient to initiate the growth of BHK cells in soft agar. Trypsin also fails to enhance the growth of a thermosensitive polyoma-transformed BHK line in soft agar at the restrictive temperature. Finally, the growth of transformed BHK cells in soft agar does not display a requirement for plasminogen and is not inhibited by soybean trypsin inhibitor. These studies argue against the involvement of plasmin or other exogenous trypsin-like enzymes in the growth and transformation of BHK cells.     

Effects of polyamine depletion on serum stimulation of quiescent 3T3 murine fibroblast cells

Numerous reports have shown that polyamines are required for cell proliferation. A current model for regulating commitment to DNA replication in cultured fibroblasts stimulated from quiescence by serum addition postulates sequential action by specific growth factors. To temporally localize polyamine-dependent steps within this defined sequence, mouse Balb/c-3T3 fibroblasts were partially depleted of polyamines by treatment with DL-alpha-difluoromethylornithine (DFMO), next rendered quiescent by serum deprivation, then stimulated by 10% serum with or without exogenous putrescine (Pu). Depletion of polyamines was verified by HPLC, and entry of cells into S phase was monitored by autoradiography. After 24 h of incubation with [3H]-thymidine, polyamine-depleted cells had labeling indices similar to quiescent cells if they were serum-stimulated without Pu, but progressed to S phase to the same degree as control cultures if polyamines were restored by adding Pu at the time of serum stimulation. These observations suggested that commitment of quiescent cells to DNA replication may require polyamines. To determine if polyamine-dependent steps occur during the pre-commitment period (up to 12 h after serum stimulation) or only in traverse of G1 (12 h to 24 h, post-commitment), polyamine-depleted quiescent cells were serum-stimulated for 12 h without Pu, then returned to low serum with Pu. Labeling indices of these cultures remained nearly as low as those of unstimulated cells. Reducing serum concentration from 10% to 0.5% at 12 h after stimulation did not effect labeling indices of control cells not depleted of polyamines by DFMO. These results supported the postulated requirement for polyamines during pre-commitment events. However, polyamine-deficient quiescent cells serum-stimulated without Pu for periods longer than 24 h had labeling indices at 36 and 48 h significantly greater than at 24 h. This suggested that polyamine depletion may decrease the rate at which quiescent cells commit to DNA replication, rather than producing an absolute blockade during the pre-commitment period.     

Dipeptidyl Peptidase 2 is an essential survival factor in the regulation of cell quiescence

Most cells in the body are in a resting state and undergo cell cycle progression only upon growth factor stimulation or activation. While much research on proliferation and activation has been performed, very little about signals that maintain quiescent cells in G₀ is known, preventing cell cycle entry or apoptosis. In this study, the pathways of apoptosis induction in quiescent peripheral blood cells and fibroblasts mediated by inhibition or down-regulation of Dipeptidyl Peptidase 2 (DPP2) have been explored. A decrease in DPP2 activity was found to cause resting cells to exit from G0, accompanied by a decrease in p130, p27^Kip1 and p21^Cip1 protein levels. In addition, DPP2-inhibited or down-regulated cells exhibit an increase in early G1/S progressors, with increases in the levels of retinoblastoma (pRb), p107 and cyclin D proteins. Furthermore, decrease of DPP2 activity leads to an increase in c-Myc and a decrease in Bcl-2, two events that have been associated with apoptosis induction. This apoptosis by DPP2 down-regulation is prevented in p53-/- cells or by ectopic expression of proteins that suppress p53 or c-Myc activity. Thus, DPP2 is essential for maintaining lymphocytes and fibroblasts in G₀, and its inhibition results in apoptosis mediated by induction of c-Myc and p53.     

Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells.

We investigated the molecular mechanisms underlying the ability of heparin to inhibit vascular smooth muscle cell (VSMC) growth. Previous experiments have shown that heparin inhibits induction of c-fos and c-myc protooncogene mRNA in rat VSMC stimulated by phorbol 12-myristate 13-acetate (PMA) but not when stimulated by epidermal growth factor (EGF) (Pukac, L. A., Castellot, J. J., Wright, T. C., Caleb, B. L., and Karnovsky, M. J. (1990) Cell Regul. 1, 435-443). The present experiments show that these mitogens activate distinct second messenger pathways in VSMC, because PMA but not EGF induction of c-fos and c-myc mRNA was suppressed in protein kinase C (PKC) down-regulated VSMC; this suggests that EGF does not act through a PKC-dependent pathway for induction of these genes. Heparin inhibited serum stimulation of c-fos mRNA in control VSMC, but heparin did not inhibit the smaller but significant serum stimulation of c-fos mRNA in PKC down-regulated VSMC, indicating that heparin may selectively inhibit PKC-dependent, but not PKC-independent, stimulation of gene expression. To further determine if heparin inhibits non-PKC pathways, VSMC were treated with dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, and Ca2+ ionophore A23187; stimulation of c-fos mRNA by this treatment was not inhibited by heparin. DNA synthesis and cell proliferation were inhibited in rat VSMC exposed briefly to heparin during the G0/G1 phase of the cell cycle. These experiments indicate heparin can act early in the cell cycle and suggest PKC-dependent but not PKC-independent signaling pathways for gene expression are selectively sensitive to heparin inhibition.     

Uncoupling of RNA and DNA synthesis after plasma stimulation of G0-arrested BALB/C-3T3 cells.

The addition of whole serum to G0-arrested, confluent Balb/c-3T3 cells induces them to progress through G1 and synthesize DNA after a 12-h lag period. Prior to the onset of DNA synthesis, RNA is synthesized and RNA content increases. Serum has been fractionated into two sets of growth factors: a platelet-derived growth factor present in heat-treated (100 degrees C) platelet extracts and platelet-poor plasma. Addition of whole serum, platelet-derived growth factor or platelet-poor plasma induces quiescent cells to increase their cytoplasmic RNA content, but the cells treated with platelet-poor plasma do not synthesize DNA. Messenger RNA content increases within 2 h after stimulation with whole serum or platelet-poor plasma, and after 18 h, mRNA has accumulated to a greater degree than rRNA.     

D-type cyclin-dependent kinase activity in mammalian cells.

D-type cyclin-dependent kinase activities have not so far been detected in mammalian cells. Lysis of rodent fibroblasts, mouse macrophages, or myeloid cells with Tween 20 followed by precipitation with antibodies to cyclins D1, D2, and D3 or to their major catalytic partner, cyclin-dependent kinase 4 (cdk4), yielded kinase activities in immune complexes which readily phosphorylated the retinoblastoma protein (pRb) but not histone H1 or casein. Virtually all cyclin D1-dependent kinase activity in proliferating macrophages and fibroblasts could be attributed to cdk4. When quiescent cells were stimulated by growth factors to enter the cell cycle, cyclin D1-dependent kinase activity was first detected in mid G1, reached a maximum near the G1/S transition, and remained elevated in proliferating cells. The rate of appearance of kinase activity during G1 phase lagged significantly behind cyclin induction and correlated with the more delayed accumulation of cdk4 and formation of cyclin D1-cdk4 complexes. Thus, cyclin D1-associated kinase activity was not detected during the G0-to-G1 transition, which occurs within the first few hours following growth factor stimulation. Rodent fibroblasts engineered to constitutively overexpress either cyclin D1 alone or cyclin D3 together with cdk4 exhibited greatly elevated cyclin D-dependent kinase activity, which remained absent in quiescent cells but rose to supraphysiologic levels as cells progressed through G1. Therefore, despite continued enforced overproduction of cyclins and cdk4, the assembly of cyclin D-cdk4 complexes and the appearance of their kinase activities remained dependent upon serum stimulation, indicating that upstream regulators must govern formation of the active enzymes.     

Specific growth stimulation of cultured smooth muscle cells from spontaneously hypertensive rats by platelet-derived growth factor A-chain homodimer.

Cultured vascular smooth muscle cells (VSMC)1 from spontaneously hypertensive rats (SHR) possess specific cell surface receptors for both homodimeric forms of platelet-derived growth factor (PDGF-AA and PDGF-BB), in contrast to cells from normotensive Wistar Kyoto (WKY) animals, which express receptors only for the B-chain form of PDGF. Stimulation of quiescent VSMC from SHR with PDGF-AA resulted in activation of S6-kinase and induction of phosphoinositide catabolism, as well as cellular proliferation when cultures were maintained for prolonged periods with daily supplementation of the growth factor. WKY-derived VSMC showed no response to PDGF-AA, which was consistent with their lack of specific receptors for this homodimer. The responsiveness of quiescent cells from SHR and WKY to the B-chain homodimer was similar. The enhanced growth responsiveness of SHR-derived cells to fetal calf serum, as compared with cells from their normotensive counterparts, may be accounted for in part by their expression of receptors for the AA homodimer of PDGF.     

G0-G1 cell cycle phase transition as revealed by fluorescence resonance energy transfer: analysis of human fibroblast chromatin

In the present study, microspectrofluorometry and digital imaging procedures were used to investigate by fluorescence Resonance Energy Transfer (FRET) analysis the changes of chromatin organization during the transition from G0 quiescent stat to G1 phase. G0 transition is a key event in cell cycle progress depending on the activation of specific genes and the concomitant silencing of others, which both entail spatial chromatin rearrangement. Normal human fibroblasts arrested in G0-phase by culture in low-serum containing medium and stimulated to re-enter G1 by serum addition were used as cell model. To investigate the occurrence and timing of these supramolecular chromatin changes, we estimated the relative FRET efficiency in single cells after double-helical DNA. Hoechst 33258 amd propidium iodide were used as a donor-acceptor dye pair since they exhibit particularly favourable spectral characteristics, that allow the calculation procedure to be semplified. The results of FRET analysis were compared to those of the immunocytochemical labelling of two nuclear proteins (i.e., Ki-67 and statin) whose expression is an established marker of potentially proliferating G1 cells or resting G0 cells, respectively. FRET efficiency was lower in G0 than G1 fibroblasts: this is likely due to higher chromatin packaging in quiescent cells which especially hinders the interaction with the donor molecules less favourable, in terms of relative distance and spatial orientation. FRET efficiency significantly increased shortly (1h) after serum stimulation of quiescent fibroblasts, thus indicating that chromatin is rearranged in parallel with activation of cycle-related gene; it is worth noting that these signs largely preceded the occurrence of immunopositivity for Ki-67, which was detectable only 24h after serum stimulation. FRET-based analyses which already proved to be suitable for studying the overall chromatin organization in differentiated cells, may now be envisaged as a powerful tool for detecting, in single cells, more subtle changes linked to the activation of early cycle-related genes.     

Uric acid stimulates vascular smooth muscle cell proliferation by increasing platelet-derived growth factor A-chain expression

Recent data suggest that uric acid is generated locally in the vessel wall by the action of xanthine oxidase. This enzyme, activated during ischemia/reperfusion by proteolytic conversion of xanthine dehydrogenase, catalyzes the oxidation of xanthine, thereby generating free radicals and uric acid. Because of the potential role of ischemia/reperfusion in vascular disease, we studied the effects of uric acid on rat aortic vascular smooth muscle cell (VSMC) growth. Uric acid stimulated VSMC DNA synthesis, as measured by [3H]thymidine incorporation, in a concentration-dependent manner with half-maximal activity at 150 microM. Maximal induction of DNA synthesis by uric acid (250 microM) was approximately 70% of 10% calf serum and equal to 10 ng/ml platelet-derived growth factor (PDGF) AB or 20 ng/ml fibroblast growth factor. Neither uric acid precursors (xanthine and hypoxanthine) nor antioxidants (ascorbic acid, glutathione, and alpha-tocopherol) were mitogenic for VSMC. Uric acid was mitogenic for VSMC but not for fibroblasts or renal epithelial cells. The time course for uric acid stimulation of VSMC growth was slower than serum, suggesting induction of an autocrine growth mechanism. Exposure of quiescent VSMC to uric acid stimulated accumulation of PDGF A-chain mRNA (greater than 5-fold at 8 h) and secretion of PDGF-like material in conditioned medium (greater than 10-fold at 24 h). Uric acid-induced [3H]thymidine incorporation was markedly inhibited by incubation with anti-PDGF A-chain polyclonal antibodies. Thus uric acid stimulates VSMC growth via an autocrine mechanism involving PDGF A-chain. These findings suggest that generation of uric acid during ischemia/reperfusion contributes to atherogenesis and intimal proliferation following arterial injury.     

Growth factor regulation of membrane transport in human fibroblasts and its relationship to stimulation of DNA synthesis

Serum stimulation of serum-deprived or density-inhibited normal cells enhances the level of various nutrient and ionic transport systems. Certain of these systems have been implicated in the regulation of cell proliferation. However, the use of serum stimulation to activate quiescent cells leads to enhancement of numerous transport systems with little understanding of which component or components of serum are related to activation of which transport systems. In this study we attempt to identify the specific effect of three known growth promoting factors (insulin, dexamethasone and epidermal growth factor [EGF]) on the activation of four membrane transport systems (A-amino acids, L-amino acids, glucose and K+) in normal and SV40-transformed WI38 human fibroblasts. We have also evaluated the effect of these growth factors on the stimulation of DNA synthesis in growth factor deprived cells. Thus, we can correlate the effect on a given transport system with the relative mitogenic stimulation produced by the growth factor. We conclude a) that a growth factor can effect a transport system differently in a normal versus transformed cell, b) that a specific growth factor can effect multiple transport systems and, c) with the exception of K+ transport, enhanced transport induced by a given growth factor does not necessarily correlate with the mitogenic potency of the growth factor. This latter point is of particular significance since the activation of K+ transport reflects, based on other studies, activation of the Na+-H+ exchanger which has been implicated in cell-cycle activation.     

Essential roles of integrin-mediated signaling for the enhancement of malignant properties of melanomas based on the expression of GD3

We reported that ganglioside GD3 enhances cell proliferation and invasion of melanomas causing stronger tyrosine-phosphorylation of p130Cas and paxillin after stimulation with fetal calf serum. Besides signals via growth factor/receptor, adhesion signals via integrin might be also enhanced by GD3. Here, roles of integrin-mediated signaling in the cell proliferation and invasion, and in the activation of adaptor molecules were examined, showing that integrin was also important for the cell growth and invasion. p130Cas and paxillin underwent stronger tyrosine-phosphorylation in GD3+ cells than in GD3− cells during the adhesion in the absence of serum. On the other hand, no proteins underwent tyrosine phosphorylation in GD3+ and GD3− cells in a suspension state when stimulated with fetal calf serum. These results suggested that integrin-mediated signaling is essential in the effects of GD3 on the malignant properties of melanomas. Co-localization of GD3 and integrin at the focal adhesion supported these results.     

Amiloride sensitive activation of S6 kinase by angiotensin II in cultured vascular smooth muscle cells

Angiotensin II was shown to activate S6-kinase in cultured vascular smooth muscle cells (VSMC) in a dose- (10(-9)-10(-6) M) and time-dependent manner. Pretreatment of quiescent cells with 12-O-Tetradecanoylphorbol-13-acetate had no effect on the activation levels of the kinase at the hormone levels used. However, stimulation of S6-kinase activity by angiotensin II was markedly inhibited by the inclusion of amiloride hydrochloride in serum-free medium during activation procedures. Angiotensin was not mitogenic for VSMC at even the highest doses used (10(-6) M). These findings support the notion that raised intracellular pH results in the activation of protein synthesis in quiescent cells.