Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis.

A phosphorothioate-oligonucleotide-based antisense strategy for depleting MAP kinase was developed. The 17mer antisense probe, EAS 1, caused a potent and concentration-dependent decrease in the steady state expression of p42 and p44 MAP kinase in 3T3 L1 fibroblasts and adipocytes with submicromolar concentrations effective. Antisense EAS 1 elicited a dose-dependent inhibition of insulin- and serum-stimulated DNA synthesis. Elimination of p42 MAP kinase by > 95% and p44 MAP kinase to levels undetected blocked the ability of serum in 3T3 L1 fibroblasts and insulin in 3T3 L1 adipocytes to stimulate DNA synthesis by 87-95%. The differentiation of 3T3 L1 fibroblasts into adipocytes was prevented by 1 microM antisense EAS 1. The corresponding sense, scrambled or sense plus antisense EAS 1 phosphorothioate oligonucleotides did not deplete the p42 or p44 MAP kinase from either cell type, did not inhibit stimulation of DNA synthesis and did not interfere with differentiation. Two kinases on different MAP kinase activation pathways were not depleted by antisense EAS 1 whereas the ability of insulin to activate p90 S6 kinase was > 90% eliminated in 3T3 L1 adipocytes by 4.5 microM antisense EAS 1. In conclusion these results show that MAP kinase is required for insulin and serum stimulation of DNA synthesis, for insulin stimulation of p90 S6 kinase activity and for differentiation of 3T3 L1 cells. Moreover, the development of the antisense probe EAS 1 against a target sequence of p42 MAP kinase that is conserved in p44 MAP kinase and across a range of species provides a molecular tool of general applicability for further dissecting the precise targets and roles of MAP kinase.     

Caveolin-1 expression sensitizes fibroblastic and epithelial cells to apoptotic stimulation

The potential role of caveolin-1 in apoptosis remains controversial. Here, we investigate whether caveolin-1 expression is proapoptotic or antiapoptotic using a well-defined antisense approach. We show that NIH/3T3 cells harboring antisense caveolin-1 are resistant to staurosporine-induced apoptosis, as assessed using cell morphology, DNA content, caspase 3 activation, and focal adhesion kinase cleavage. Importantly, sensitivity to apoptosis is recovered when caveolin-1 levels are restored. Conversely, recombinant stable expression of caveolin-1 in T24 bladder carcinoma cells sensitizes these cells to caspase 3 activation. Consistent with the observations using NIH/3T3 cells, downregulation of caveolin-1 in T24 cells substantially diminishes caspase 3-like activity. Loss of sensitivity to apoptotic stimulation is recovered by inhibition of the phosphatidylinositol 3-kinase pathway using LY-294002, suggesting a possible mechanism for the sensitizing effect of caveolin-1. Thus our results suggest that caveolin-1 may act as a coupling or sensitizing factor in signaling apoptotic cell death in both fibroblastic (NIH/3T3) and epithelial (T24) cells.     

Caveolin-1 inhibits epidermal growth factor-stimulated lamellipod extension and cell migration in metastatic mammary adenocarcinoma cells (MTLn3). Transformation suppressor effects of adenovirus-mediated gene delivery of caveolin-1

Caveolin-1 is a principal component of caveolae membranes that may function as a transformation suppressor. For example, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (D7S522; 7q31.1) that is deleted in human cancers, including mammary carcinomas. However, little is known about the role of caveolins in regulating cell movement, a critical parameter in determining metastatic potential. Here, we examine the role of caveolin-1 in cell movement. For this purpose, we employed an established cellular model, MTLn3, a metastatic rat mammary adenocarcinoma cell line. In this system, epidermal growth factor (EGF) stimulation induces rapid lamellipod extension and cell migration. Interestingly, we find that MTLn3 cells fail to express detectable levels of endogenous caveolin-1. To restore caveolin-1 expression in MTLn3 cells efficiently, we employed an inducible adenoviral gene delivery system to achieve tightly controlled expression of caveolin-1. We show here that caveolin-1 expression in MTLn3 cells inhibits EGF-stimulated lamellipod extension and cell migration and blocks their anchorage-independent growth. Under these conditions, EGF-induced activation of the p42/44 mitogen-activated protein kinase cascade is also blunted. Our results suggest that caveolin-1 expression in motile MTLn3 cells induces a non-motile phenotype.     

p42/p44 MAP kinase activation is localized to caveolae-free membrane domains in airway smooth muscle

Caveolae are abundant plasma membrane invaginations in airway smooth muscle that may function as preorganized signalosomes by sequestering and regulating proteins that control cell proliferation, including receptor tyrosine kinases (RTKs) and their signaling effectors. We previously demonstrated, however, that p42/p44 MAP kinase, a critical effector for cell proliferation, does not colocalize with RTKs in caveolae of quiescent airway myocytes. Therefore, we investigated the subcellular sites of growth factor-induced MAP kinase activation. In quiescent myocytes, though epidermal growth factor receptor (EGFR) was almost exclusively found in caveolae, p42/p44 MAP kinase, Grb2, and Raf-1 were absent from these membrane domains. EGF induced concomitant phosphorylation of caveolin-1 and p42/p44 MAP kinase; however, EGF did not promote the localization of p42/p44 MAP kinase, Grb2, or Raf-1 to caveolae. Interestingly, stimulation of muscarinic M(2) and M(3) receptors that were enriched in caveolae-deficient membranes also induced p42/p44 MAP kinase phosphorylation, but this occurred in the absence of caveolin-1 phosphorylation. This suggests that the localization of receptors to caveolae and interaction with caveolin-1 is not directly required for p42/p44 MAP kinase phosphorylation. Furthermore, we found that EGF exposure induced rapid translocation of EGFR from caveolae to caveolae-free membranes. EGFR trafficking coincided temporally with EGFR and p42/p44 MAP kinase phosphorylation. Collectively, this indicates that although caveolae sequester some receptors associated with p42/p44 MAP kinase activation, the site of its activation is associated with caveolae-free membrane domains. This reveals that directed trafficking of plasma membrane EGFR is an essential element of signal transduction leading to p42/p44 MAP kinase activation.     

Cellular stress induces the tyrosine phosphorylation of caveolin-1 (Tyr(14)) via activation of p38 mitogen-activated protein kinase and c-Src kinase. Evidence for caveolae, the actin cytoskeleton, and focal adhesions as mechanical sensors of osmotic stress

Environmental stressors have been recently shown to activate intracellular mitogen-activated protein (MAP) kinases, such as p38 MAP kinase, leading to changes in cellular functioning. However, little is known about the downstream elements in these signaling cascades. In this study, we show that caveolin-1 is phosphorylated on tyrosine 14 in NIH 3T3 cells after stimulation with a variety of cellular stressors (i.e. high osmolarity, H2O2, and UV light). To detect this phosphorylation event, we employed a phosphospecific monoclonal antibody probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Since p38 MAP kinase and c-Src have been previously implicated in the stress response, we next assessed their role in the tyrosine phosphorylation of caveolin-1. Interestingly, we show that the p38 inhibitor (SB203580) and a dominant-negative mutant of c-Src (SRC-RF) both block the stress-induced tyrosine phosphorylation of caveolin-1 (Tyr(P)(14)). In contrast, inhibition of the p42/44 MAP kinase cascade did not affect the tyrosine phosphorylation of caveolin-1. These results indicate that extracellular stressors can induce caveolin-1 tyrosine phosphorylation through the activation of well established upstream elements, such as p38 MAP kinase and c-Src kinase. However, heat shock did not promote the tyrosine phosphorylation of caveolin-1 and did not activate p38 MAP kinase. Finally, we show that after hyperosmotic shock, tyrosine-phosphorylated caveolin-1 is localized near focal adhesions, the major sites of tyrosine kinase signaling. In accordance with this localization, disruption of the actin cytoskeleton dramatically potentiates the tyrosine phosphorylation of caveolin-1. Taken together, our results clearly define a novel signaling pathway, involving p38 MAP kinase activation and caveolin-1 (Tyr(P)(14)). Thus, tyrosine phosphorylation of caveolin-1 may represent an important downstream element in the signal transduction cascades activated by cellular stress.     

Expression of caveolin-1 induces premature cellular senescence in primary cultures of murine fibroblasts

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-1 is the principal structural component of caveolae in vivo. Several lines of evidence are consistent with the idea that caveolin-1 functions as a "transformation suppressor" protein. In fact, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). We have previously demonstrated that overexpression of caveolin-1 arrests mouse embryonic fibroblasts in the G(0)/G(1) phase of the cell cycle through activation of a p53/p21-dependent pathway, indicating a role of caveolin-1 in mediating growth arrest. However, it remains unknown whether overexpression of caveolin-1 promotes cellular senescence in vivo. Here, we demonstrate that mouse embryonic fibroblasts transgenically overexpressing caveolin-1 show: 1) a reduced proliferative lifespan; 2) senescence-like cell morphology; and 3) a senescence-associated increase in beta-galactosidase activity. These results indicate for the first time that the expression of caveolin-1 in vivo is sufficient to promote and maintain the senescent phenotype. Subcytotoxic oxidative stress is known to induce premature senescence in diploid fibroblasts. Interestingly, we show that subcytotoxic level of hydrogen peroxide induces premature senescence in NIH 3T3 cells and increases endogenous caveolin-1 expression. Importantly, quercetin and vitamin E, two antioxidant agents, successfully prevent the premature senescent phenotype and the up-regulation of caveolin-1 induced by hydrogen peroxide. Also, we demonstrate that hydrogen peroxide alone, but not in combination with quercetin, stimulates the caveolin-1 promoter activity. Interestingly, premature senescence induced by hydrogen peroxide is greatly reduced in NIH 3T3 cells harboring antisense caveolin-1. Importantly, induction of premature senescence is recovered when caveolin-1 levels are restored. Taken together, these results clearly indicate a central role for caveolin-1 in promoting cellular senescence and they suggest the hypothesis that premature senescence may represent a tumor suppressor function mediated by caveolin-1 in vivo.     

Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase.

We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid. This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade. Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor. This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.     

Caveolin-1 null mice develop cardiac hypertrophy with hyperactivation of p42/44 MAP kinase in cardiac fibroblasts

Recently, development ofa caveolin-1-deficient (Cav-1 null) mouse model has allowed thedetailed analysis of caveolin-1's function in the context of awhole animal. Interestingly, we now report that the hearts ofCav-1 null mice are markedly abnormal, despite the fact that caveolin-1is not expressed in cardiac myocytes. However, caveolin-1 is abundantlyexpressed in the nonmyocytic cells of the heart, i.e., cardiacfibroblasts and endothelia. Quantitative imaging studies of Cav-1 nullhearts demonstrate a significantly enlarged right ventricular cavityand a thickened left ventricular wall with decreased systolic function.Histological analysis reveals myocyte hypertrophy withinterstitial/perivascular fibrosis. Because caveolin-1 is thought toact as a negative regulator of the p42/44 MAP kinase cascade, weperformed Western blot analysis with phospho-specific antibodies thatonly recognize activated ERK1/2. As predicted, the p42/44 MAP kinasecascade is hyperactivated in Cav-1 null heart tissue (i.e.,interstitial fibrotic lesions) and isolated cardiac fibroblasts. Inaddition, endothelial and inducible nitric oxide synthase levels aredramatically upregulated. Thus loss of caveolin-1 expression drivesp42/44 MAP kinase activation and cardiac hypertrophy.

     

Cyclic AMP-mediated inhibition of cell growth requires the small G protein Rap1

In many normal and transformed cell types, the intracellular second messenger cyclic AMP (cAMP) blocks the effects of growth factors and serum on mitogenesis, proliferation, and cell cycle progression. cAMP exerts these growth-inhibitory effects via inhibition of the mitogen-activated protein (MAP) kinase cascade. Here, using Hek293 and NIH 3T3 cells, we show that cAMP's inhibition of the MAP kinase cascade is mediated by the small G protein Rap1. Activation of Rap1 by cAMP induces the association of Rap1 with Raf-1 and limits Ras-dependent activation of ERK. In NIH 3T3 cells, Rap1 is required not only for cAMP's inhibition of ERK activation but for inhibition of cell proliferation and mitogenesis as well.     

Ethanol potentiates the mitogenic effects of sphingosine 1-phosphate by a zinc- and calcium-dependent mechanism in fibroblasts.

In mouse embryo NIH 3T3 fibroblasts, ethanol (60-80 mM) was found to enhance the stimulatory effects of sphingosine 1-phosphate (S1P) on both DNA synthesis and cell proliferation. Well-detectable potentiating effects of ethanol on S1P-induced mitogenesis required the presence of calcium (>1 mM) and zinc (20-40 microM) in the incubation medium. The amphibian tetrapeptide bombesin, which is known to mobilize intracellular calcium in fibroblasts, had no effect alone, but it approximately doubled the combined stimulatory effects of ethanol and S1P on DNA synthesis. The synergistic mitogenic effects of ethanol and S1P were also slightly enhanced, rather than inhibited, by the alcohol dehydrogenase inhibitor 4-methylpyrazole (5 mM). Of the various growth regulatory enzymes examined, ethanol detectably enhanced the stimulatory effects of S1P on the phosphosphorylation (activation) of p42/p44 mitogen-activated protein (MAP) kinases, but not of p38 MAP kinase. Cotreatment of fibroblasts with ethanol for 10 min also enhanced the stimulatory effects of S1P on the activities of c-Raf-1 kinase and p70 S6 kinase, but neither S1P nor ethanol had effects on phosphatidylinositol 3'-kinase and Akt/PKB kinase activities. Ethanol-plus-S1P-induced DNA synthesis was partially inhibited by both PD 98059 (50 microM) and rapamycin (10 nM), inhibitors of p42/p44 MAP kinase kinase and mTOR/p70 S6 kinases, respectively. The results indicate that in NIH 3T3 fibroblasts, ethanol can enhance the mitogenic effects of S1P by a zinc- and calcium-dependent mechanism involving both the rapamycin-sensitive p70 S6 kinase-dependent and the c-Raf-1/MAP kinase-dependent growth regulatory pathways.     

Targeted down-regulation of caveolin-3 is sufficient to inhibit myotube formation in differentiating C2C12 myoblasts. Transient activation of p38 mitogen-activated protein kinase is required for induction of caveolin-3 expression and subsequent myotube formation.

Caveolin-3 is the principal structural protein of caveolae membrane domains in striated muscle cells. Caveolin-3 mRNA and protein expression are dramatically induced during the differentiation of C2C12 skeletal myoblasts, coincident with myoblast fusion. In these myotubes, caveolin-3 localizes to the sarcolemma (muscle cell plasma membrane), where it associates with the dystrophin-glycoprotein complex. However, it remains unknown what role caveolin-3 plays in myoblast differentiation and myotube formation. Here, we employ an antisense approach to derive stable C2C12 myoblasts that fail to express the caveolin-3 protein. We show that C2C12 cells harboring caveolin-3 antisense undergo differentiation and express normal amounts of four muscle-specific marker proteins. However, C2C12 cells harboring caveolin-3 antisense fail to undergo myoblast fusion and, therefore, do not form myotubes. Interestingly, treatment with specific p38 mitogen-activated protein kinase inhibitors blocks both myotube formation and caveolin-3 expression, but does not affect the expression of other muscle-specific proteins. In addition, we find that three human rhabdomyosarcoma cell lines do not express caveolin-3 and fail to undergo myoblast fusion. Taken together, these results support the idea that caveolin-3 expression is required for myoblast fusion and myotube formation, and suggest that p38 is an upstream regulator of caveolin-3 expression.     

The p42/p44 mitogen-activated protein kinase activation triggers p27Kip1 degradation independently of CDK2/cyclin E in NIH 3T3 cells.

The p42/p44 mitogen-activated protein (MAP) kinase is stimulated by various mitogenic stimuli, and its sustained activation is necessary for cell cycle G(1) progression and G(1)/S transition. G(1) progression and G(1)/S transition also depend on sequential cyclin-dependent kinase (CDK) activation. Here, we demonstrate that MAP kinase inhibition leads to accumulation of the CDK inhibitor p27(Kip1) in NIH 3T3 cells. Blocking the proteasome-dependent degradation of p27(Kip1) impaired this accumulation, suggesting that MAP kinase does not act on p27(Kip1) protein synthesis. In the absence of extracellular signals (growth factors or cell adhesion), genetic activation of MAP kinase decreased the expression of p27(Kip1) as assessed by cotransfection experiments and by immunofluorescence detection. Importantly, MAP kinase activation also decreased the expression of a p27(Kip1) mutant, which cannot be phosphorylated by CDK2, suggesting that MAP kinase-dependent p27(Kip1) regulation is CDK2-independent. Accordingly, expression of dominant-negative CDK2 did not impair the down-regulation of p27(Kip1) induced by MAP kinase activation. These data demonstrate that the MAP kinase pathway regulates p27(Kip1) expression in fibroblasts essentially through a degradation mechanism, independently of p27(Kip1) phosphorylation by CDK2. This strengthens the role of this CDK inhibitor as a key effector of G(1) growth arrest, whose expression can be controlled by extracellular stimuli-dependent signaling pathways.     

Platelet-derived growth factor-BB amplifies PGF2alpha-stimulated VEGF synthesis in osteoblasts: function of phosphatidylinositol 3-kinase

We have reported that prostaglandin F2alpha (PGF2alpha) stimulates the synthesis of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In addition, we recently showed that phosphatidylinositol 3 (PI3)-kinase activated by platelet-derived growth factor-BB (PDGF-BB) negatively regulates the interleukin-6 synthesis in these cells. In the present study, we investigated the effect of PDGF-BB on the PGF2alpha-induced VEGF synthesis in MC3T3-E1 cells. PDGF-BB, which alone did not affect the levels of VEGF, significantly enhanced the PGF2alpha-stimulated VEGF synthesis. The amplifying effect of PDGF-BB was dose dependent in the range between 10 and 70 ng/ml. LY294002 or wortmannin, specific inhibitors of PI3-kinase, which by itself failed to affect the PGF2alpha-stimulated VEGF synthesis, significantly suppressed the amplification by PDGF-BB. PD98059, a specific inhibitor of MEK1/2, suppressed the amplification by PDGF-BB of the PGF2alpha-stimulated VEGF synthesis similar to the levels of PGF2alpha with PD98059. PDGF-BB itself induced the phosphorylation of p44/p42 MAP kinase in these cells, and the effects of PDGF-BB and PGF2alpha on the phosphorylation of p44/p42 MAP kinase were additive. Moreover, LY294002 had little effect on the phosphorylation of p44/p42 MAP kinase induced by PGF2alpha with PDGF-BB. These results strongly suggest that PGF2alpha-stimulated VEGF synthesis is amplified by PI3-kinase-mediating PDGF-BB signaling in osteoblasts, and that the effect is exerted at a point downstream from p44/p42 MAP kinase.     

Combined loss of INK4a and caveolin-1 synergistically enhances cell proliferation and oncogene-induced tumorigenesis: role of INK4a/CAV-1 in mammary epithelial cell hyperplasia

Tumorigenesis is a multistep process that involves a series of genetic changes or "multiple hits," leading to alterations in signaling, proliferation, immortalization, and transformation. Many of the molecular factors that govern tumor initiation and progression remain unknown. Here, we evaluate the transformation suppressor potential of caveolin-1 (Cav-1) and its ability to cooperate with a well established tumor suppressor, the INK4a locus. To study the effects of loss of caveolin-1 on cellular transformation, we established immortalized primary mouse embryonic fibroblasts (MEFs) expressing and lacking caveolin-1 by interbreeding Cav-1 (+/+) and Cav-1 (-/-) mice with INK4a (-/-) mice. Analysis of these cells reveals that loss of caveolin-1 confers a significant growth advantage, as measured via cellular proliferation and cell cycle analysis. Loss of caveolin-1 in the INK4a (-/-) genetic background results in constitutive hyperactivation of the p42/44 MAP kinase cascade, decreased expression of p21(Cip1), as well as cyclin D1 and PCNA overexpression, consistent with their hyperproliferative phenotype. Importantly, in cells lacking Cav-1 expression, transformation by activated oncogenes (H-Ras(G12V) or v-Src) results in increased tumor growth in vivo (up to >40-fold). Finally, INK4a (-/-)/Cav-1 (-/-) mice demonstrate disturbed mammary epithelial ductal morphology, with hyperplasia, increased side-branching, and fibrosis. Our results provide important new evidence for the transformation suppressor properties of Cav-1 and the first molecular genetic evidence that Cav-1 cooperates with a tumor suppressor, namely the INK4a genetic locus.     

Activation of p44/p42 mitogen-activated protein kinase limits triiodothyronine-stimulated alkaline phosphatase activity in osteoblasts

It has been shown that thyroid hormone stimulates the activity of alkaline phosphatase, a marker of mature osteoblast phenotype, in osteoblasts. In the present study, we investigated whether p44/p42 mitogen-activated protein (MAP) kinase is involved in the thyroid hormone-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. Triiodothyronine (T(3)) markedly induced the phosphorylation of p44/p42 MAP kinase. PD98059 and U0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly enhanced the T(3)-induced alkaline phosphatase activity in a dose-dependent manner. The phosphorylation of p44/p42 MAP kinase induced by T(3) was reduced by U0126. These results strongly suggest that p44/p42 MAP kinase takes part in the thyroid hormone-stimulated alkaline phosphatase activity in osteoblasts and that p44/p42 MAP kinase plays an inhibitory role in the thyroid hormone-effect.