Protein kinase A suppresses the differentiation of 3T3-L1 preadipocytes

cAMP and protein kinase A （PKA） are widely known as signaling molecules that are important for the induction of adipogenesis. Here we show that a strong increase in the amount of cAMP inhibits the adipogenesis of 3T3-L1 fibroblast cells. Stimulation of PKA activity suppresses adipogenesis and, in contrast, inhibition of PKA activity markedly accelerates the adipogenic process. As adipogenesis progresses, there is a significant increase in the expression level of PKA regulatory and a corresponding decrease in PKA activity. Moreover, treatment of 3T3-L1 cells with epidermal growth factor （EGF） stimulates PKA activity and blocks adipogenesis. Inhibition of PKA activity abolishes this suppressive effect of EGF on adipogenesis. Moreover, asubunits ctivation of PKA induces serine/threonine phosphorylation, reduces tyrosine phosphorylation of insulin receptor substrate 1 （IRS-1） and the association between PKA and IRS-1. Taken together, our study demonstrates that PKA has a pivotal role in the suppression of adipogenesis, cAMP at high concentrations can suppress adipogenesis through PKA activation. These findings could be important and useful for understanding the mechanisms of adipogenesis and the relevant physiological events.     

PKCdelta-dependent cleavage and nuclear translocation of annexin A1 by phorbol 12-myristate 13-acetate.

Annexin A1 (ANX-1), a calcium-dependent, phospholipid binding protein, is known to be involved in diverse cellular processes, including regulation of cell growth and differentiation, apoptosis, and inflammation. The mitogen phorbol 12-myristate 13-acetate (PMA) induces expression and phosphorylation of ANX-1. However, the roles of ANX-1 in PMA-induced signal transduction is unknown. Here, we study the cellular localization of ANX-1 in the PMA-induced signal transduction process. We have found that PMA induces the cleavage of ANX-1 in human embryonic kidney (HEK) 293 cells, and that the cleaved form of ANX-1 translocates to the nucleus. The PMA-induced nuclear translocation of ANX-1 was inhibited by the protein kinase C (PKC)delta-specific inhibitor rottlerin, indicating that PKCdelta plays a role in nuclear translocation of the cleaved ANX-1. We propose a novel mechanism of PMA-induced translocation of ANX-1 to the nucleus that may participate in the regulation of cell proliferation and differentiation.     

Fate of immunoprecipitable protein kinase C in GH3 cells treated with phorbol 12-myristate 13-acetate

The effect of phorbol 12-myristate 13-acetate (PMA) on protein kinase C was studied by metabolically labeling GH3 cells with [35S]methionine and using a polyclonal antibody raised against rat brain protein kinase C to immunoprecipitate the enzyme. PMA accelerates the loss of immunologically reactive protein kinase C from the cells in a time- and dose-dependent manner. The half-life of the enzyme in cells treated with 400 nM PMA was 2 h whereas in control cells 60-70% of the enzyme was still detectable after 24 h. The concentration of PMA required to reduce cellular protein kinase C 50% after 24 h was 130 nM. PMA also induced the translocation of [35S]Met-labeled protein kinase C from the cytosol to the membranes in a concentration-dependent manner. Less protein kinase C was translocated to membranes when cells were treated with 20 nM PMA than when they were exposed to 400 nM PMA. In the latter case, most of the labeled protein kinase C became membrane-associated. Maximal translocation was evident after 15 min of incubation with either concentration of PMA and was followed by degradation of the membrane-associated enzyme. The rate of degradation of membrane-associated protein kinase C was the same with both concentrations of PMA. In cells treated with 20 nM PMA, disappearance of [35S]Met-labeled protein kinase C from the cytosolic fraction occurred in two phases, a rapid decrease characteristic of the membrane-associated enzyme, followed by a slower loss similar to that seen in control cells. The results indicate that turnover of protein kinase C is enhanced by membrane association.     

Loss of choleragen receptors and ganglioside upon differentiation of 3T3-L1 preadipocytes

3T3-L1 preadipocytes differentiate in culture into cells having the enzymatic and morphological characteristics of adipocytes. Differentiation is accompanied by a decrease in total cellular ganglioside content; the ganglioside level is 1.8 to 2.5-fold higher in undifferentiated than in differentiated cells. Gangliosides GM3 and GD1a constitute a majority of total cell gangliosides in both cell types, while ganglioside GM1, the putative choleragen receptor, constitutes less than 5%. Differentiation results in a 75 to 85% decrease in ganglioside GM1. An inverse correlation exists between the percentage of adipocytes in the cell population and: 1) total ganglioside and ganglioside GM1 content, and 2) surface ganglioside GM1 as estimated by choleragen binding or fluorescent staining of bound choleragen. Nondifferentiating 3T3-C2 control cells do not exhibit changes in total ganglioside, ganglioside GM1, or choleragen binding that are observed with 3T3-L1 cells.     

PKC-dependent activation of sphingosine kinase 1 and translocation to the plasma membrane. Extracellular release of sphingosine-1-phosphate induced by phorbol 12-myristate 13-acetate (PMA)

Sphingosine-1-phosphate (S1P) is a highly bioactive sphingolipid involved in diverse biological processes leading to changes in cell growth, differentiation, motility, and survival. S1P generation is regulated via sphingosine kinase (SK), and many of its effects are mediated through extracelluar action on G-protein-coupled receptors. In this study, we have investigated the mechanisms regulating SK, where this occurs in the cell, and whether this leads to release of S1P extracellularly. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), induced early activation of SK in HEK 293 cells, and this activation was more specific to the membrane-associated SK. Therefore, we next investigated whether PMA induced translocation of SK to the plasma membrane. PMA induced translocation of both endogenous and green fluorescent protein (GFP)-tagged human SK1 (hSK1) to the plasma membrane. PMA also induced phosphorylation of GFP-hSK1. The PMA-induced translocation was abrogated by preincubation with known PKC inhibitors (bisindoylmaleimide and calphostin-c) as well as by the indirect inhibitor of PKC, C(6)-ceramide, supporting a role for PKC in mediating translocation of SK to the plasma membrane. SK activity was not necessary for translocation, because a dominant negative G82D mutation also translocated in response to PMA. Importantly, PKC regulation of SK was accompanied by a 4-fold increase in S1P in the media. These results demonstrate a novel mechanism by which PKC regulates SK and increases secretion of S1P, allowing for autocrine/paracrine signaling.     

Role of protein kinase C activation in oocyte maturation and steroidogenesis in ovarian follicles of Rana pipiens: Studies with phorbol 12-myristate 13-acetate

In ovarian follicles of Rana pipiens, frog pituitary homogenates (FPH) elevate intrafollicular progesterone levels which in turn is thought to induce meiotic resumption in the prophase I arrested oocytes. Calcium plays a role in FPH and steroid-provoked responses in the somatic and gametic components of the follicle, presumably via effects exerted at the plasma membrane of their respective target cells. Many membrane active hormones which utilize Ca²⁺ in their intracellular transduction also provoke membrane phosphoinositide hydrolysis yielding inositol triphosphate (IP₃) and diacyl glycerol (DAG), an activator of the CA²⁺-dependent protein kinase C (PKC). The actions of phorbol 12-myristate 13-acetate (TPA), a potent synthetic activator of PKC, on progesterone production and oocyte maturation was examined in in vitro cultured ovarian follicles. TPA induced germinal vesicle breakdown (GVBD) in intact follicles and in oocytes denuded of somatic components, while the inactive compound phorbol 13-monoacetate was ineffective. Further, TPA induction of GVBD exhibited similarities to progesterone-induced GVBD, being inhibited by treatments which elevate cAMP or inhibit protein synthesis. TPA alone did not elevate intrafollicular or medium progesterone levels, as occurred in FPH-treated follicles. TPA partially inhibited intrafollicular progesterone accumulation induced by FPH or treatments which elevate cAMP levels. These data suggest that activation of PKC plays a role in oocyte maturation independent of follicular progesterone production as occurs in response to FPH. Further, it appears that the somatic cells of the amphibian follicle also possess PKC which when activated, antagonizes cAMP generating pathway in these cells. Results indicate that protein kinase can influence oocyte maturation in Rana follicular oocytes by several mechanisms.     

CD45 negatively regulates monocytic cell differentiation by inhibiting phorbol 12-myristate 13-acetate-dependent activation and tyrosine phosphorylation of protein kinase Cdelta

The protein-tyrosine phosphatase CD45 is expressed on all monocytic cells, but its function in these cells is not well defined. Here we report that CD45 negatively regulates monocyte differentiation by inhibiting phorbol 12-myristate 13-acetate (PMA)-dependent activation of protein kinase C (PKC) delta. We found that antisense reduction of CD45 in U937 monocytic cells (CD45as cells) increased by 100% the ability of PMA to enlarge cell size, increase cell cytoplasmic process width and length, and induce surface expression of CD11b. In addition, reduction in CD45 expression caused the duration of peak PMA-induced MEK and extracellular signal-regulated kinase (ERK) 1/2 activity to increase from 5 min to 30 min while leading to a 4-fold increase in PMA-dependent PKCdelta activation. Importantly, PMA-dependent tyrosine phosphorylation of PKCdelta was also increased 4-fold in CD45as cells. Finally, inhibitors of MEK (PD98059) and PKCdelta (rottlerin) completely blocked PMA-induced monocytic cell differentiation. Taken together, these data indicate that CD45 inhibits PMA-dependent PKCdelta activation by impeding PMA-dependent PKCdelta tyrosine phosphorylation. Furthermore, this blunting of PKCdelta activation leads to an inhibition of PKCdelta-dependent activation of ERK1/2 and ERK1/2-dependent monocyte differentiation. These findings suggest that CD45 is a critical regulator of monocytic cell development.     

Extracellular signal-regulated kinase/90-KDA ribosomal S6 kinase/nuclear factor-kappa B pathway mediates phorbol 12-myristate 13-acetate-induced megakaryocytic differentiation of K562 cells

Two signaling pathways, the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)-dependent pathway and the nuclear factor-kappaB (NF-kappaB)-dependent pathway, have been known to mediate megakaryocytic differentiation of K562 cells induced by phorbol 12-myristate 13-acetate (PMA). In this study, we examined whether 90-kDa ribosomal S6 kinase (RSK), known as a substrate of ERK/MAPK and a signal-inducible IkappaBalpha kinase, would link two pathways during the differentiation. RSK1 was activated in a time- and dose-dependent manner during the PMA-induced differentiation. Overexpression of wild-type or dominant inhibitory mutant (D205N) of RSK1 enhanced or suppressed PMA-stimulated NF-kappaB activation and megakaryocytic differentiation as shown by morphology, nonspecific esterase activity, and expression of the CD41 megakaryocytic marker, respectively. In addition, overexpression of the dominant inhibitory mutant (S32A/S36A) of IkappaBalpha inhibited PMA-stimulated and RSK1-enhanced megakaryocytic differentiation, indicating that NF-kappaB mediates a signal for megakaryocytic differentiation downstream of RSK1. PMA-stimulated activation of ERK/MAPK, RSK1, and NF-kappaB and the PMA-induced megakaryocytic differentiation were prevented by pretreatment with PD98059, a specific inhibitor of the mitogen-activated ERK kinase (MEK). Therefore, these results demonstrate that the sequential ERK/RSK1/NF-kappaB pathway mediates PMA-stimulated megakaryocytic differentiation of K562 cells.     

Protein kinase C β1 overexpression augments phorbol ester-induced increase in endothelial permeability

We studied the postulated involvement of the protein kinase C β₁ (PKCβ₁) isoform in the regulation of endothelial permeability using human dermal microvascular endothelial cell line (HMEC-1). We overexpressed the recombinant PKCβ₁ gene via retroviral-mediated transduction in these cells. PKCβ₁ gene transfer was stable, and PKCβ₁ protein production was persistent for at least 1 month posttransduction. Addition of 2 × 10⁻⁹ M and 2 × 10⁻⁸ M phorbol 12-myristate 13-acetate (PMA) to the control (nontransduced) HMEC-1 cells increased the transendothelial ¹²⁵I-albumin clearance rate (an index of endothelial permeability) from 2.5 ± 0.2 × 10⁻² μl/min to 5.4 ± 1.2 × 10⁻² μl/min and 16.8 ± 3.1 × 10⁻² μl/min, respectively. However, addition of 2 × 10⁻⁹ M PMA to PKCβ₁-overexpressing HMEC-1 cells produced a maximal increase in the transendothelial ¹²⁵I-albumin clearance rate of 15.9 ± 2.0 × 10⁻² μl/min. Challenge of these cells with 2 × 10 ⁻⁸ M PMA did not further augment the increase in permeability. Activation with PMA was associated with the translocation of the PKCβ₁ from the cytosol to the membrane. These data show that PKCβ₁ overexpression augments the increase in endothelial permeability in response to PKC activation, suggesting an important function for the PKCβ₁ isoform in the regulation of endothelial barrier. © 1996 Wiley-Liss, Inc.     

Uneven distribution of protein kinase C-α and -β isozymes in human sarcomas and carcinomas

Protein kinase C (PKC) represents a family of structurally related Ser/Tre kinases which are involved in mitogenic signalling and may contribute to human neoplasia. To address this issue, the messenger RNA and protein levels of PKC isoenzymes α and β were analyzed in several human sarcoma- and carcinoma-derived cell lines. Carcinomas contained low or undetectable levels of either PKC-α or PKC-β. Sarcomas exhibited similar or increased PKC expression compared to human diploid fibroblasts. Moreover, sarcoma cell lines expressing one PKC isoform did not contain detectable levels of the other. When PKC was depleted from the tumor cells, we observed that the PKC overexpressing sarcomas had reduced their malignant properties as determined by their ability to grow in semisolid medium. In addition, epidermal growth factor-stimulated and erbB2-transformed fibroblasts exhibited enhanced cell growth in the absence of PKC. We propose a model for the effect of PKC as a negative regulator of proliferation in epithelial cells and a growth promoter in fibroblasts. © 1994 wiley-Liss, Inc.     

Phorbol 12-myristate 13-acetate (TPA) blocks CD3-mediated Ca2+ mobilization in Jurkat T cells independently of protein kinase C activation

Stimulation of Jurkat T cells with antibodies against the T cell receptor/CD3 complex induces a rise in the intracellular concentration of Ca2+ within seconds. The inositol phosphate-dependent Ca2+ mobilization induced by OKT3 was completely abrogated when Jurkat cells were pretreated for 1 min with the phorbol 12-myristate 13-acetate TPA (10nM), a concentration which activates protein kinase C (PKC). The effects of TPA on the Ca2+ fluxes were insensitive to treatment of the cells with known PKC inhibitors (H-7 and staurosporin) under conditions where the PKC-mediated phosphorylation was blocked. Furthermore, another activator of PKC, mezerein, inhibited the Ca2+ signal induced by OKT3. This inhibition, however, could completely be reversed by pretreatment with H-7 or staurosporine. We conclude that the TPA-mediated inhibition of Ca2+ fluxes in Jurkat T cells largely acts through a PKC-independent pathway.     

Orphan nuclear receptor Nur77 accelerates the initial phase of adipocyte differentiation in 3T3-L1 cells by promoting mitotic clonal expansion

Nur77 is an orphan member of the nuclear receptor superfamily that is expressed in various types of cells and mediates diverse biological processes. Although Nur77 mRNA is induced in the early stage of adipogenesis of 3T3-L1 cells, its roles are not known. To address this issue, we closely inspected the expression of Nur77 mRNA and protein during differentiation of 3T3-L1 cells. Nur77 was induced rapidly and transiently at both mRNA and protein levels only in the initial phase of differentiation induction, and localized almost exclusively in the nuclei. Isobutylmethylxanthine was essential for the induction of Nur77 protein, acting by at least in part protecting the protein from rapid degradation by proteasome. Nur77 siRNA resulted in delayed adipogenesis in 3T3-L1, accompanied by retarded mitotic clonal expansion. These effects were mediated at least partly by decreased expression of cyclins D and E. Constitutive expression of Nur77 inhibited adipogenesis of 3T3-L1, accompanied by enhanced expression of cyclin D1 and prolonged mitotic clonal expansion. Moreover, constitutive expression of Nur77 inhibited, but transient induction of Nur77 promoted, adipogenesis in NIH-3T3 cells. These results suggest that Nur77 accelerates adipocyte differentiation by regulating cell cycle progression and the rapid and transient induction is crucial for its action.     

Phorbol ester activation of functional rat protein kinase C β-1 causes phenotype in yeast

The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of various eukaryotic cellular signals. The molecular dissection of its mechanisms of action has been limited in part by the genetic inaccessibility and complexity of signaling in mammalian cells. Here we present a novel approach to study rat PKC β-1 action in yeast, a simple lower eukaryotic genetic model. Expression of its cDNA in Saccharomyces cerevisiae introduces novel phorbol ester binding sites which stimulate a specific calcium- and phospholipid-dependent catalytic activity in vitro consistent with a fully functional protein which phosphorylates cellular yeast proteins in vivo. Phorbol ester activation of PKC β-1 in vivo results in biological responses which include stimulation of extracellular calcium uptake, changes in cell morphology, and an increase in the cell doubling time. These PKC functions are not affected by truncation of 12 amino terminal amino acids; however, they are completely abolished by truncation of 15 or more carboxyl terminal amino acids which likely result in inactivation of the kinase. The increase in the yeast doubling time caused by PKC β-1 activation provides a phenotype which can be exploited as a screen for the activity of random PKC cDNA mutations. Our findings indicate that rat PKC β-1 is functional in yeast and leads to biological responses which suggest compatible aspects of higher and lower eukaryotic signaling pathways and the feasibility of dissecting parts of the action of common signaling mediators in a simple genetic model.