Angiotensin II stimulates DNA synthesis of rat pancreatic stellate cells by activating ERK through EGF receptor transactivation

Although angiotensin II (Ang II) is known to participate in pancreatic fibrosis, little is known as to the mechanism by which Ang II promotes pancreatic fibrosis. To elucidate the mechanism, we examined the action of Ang II on the proliferation of rat pancreatic stellate cells (PSCs) that play central roles in pancreatic fibrosis. Immunocytochemistry and Western blotting demonstrated that both Ang II type 1 and type 2 receptors were expressed in PSCs. [3H]Thymidine incorporation assay revealed that Ang II enhanced DNA synthesis in PSCs, which was blocked by Ang II type 1 receptor antagonist losartan. Western blotting using anti-phospho-epidermal growth factor (EGF) receptor and anti-phospho-extracellular signal regulated kinase (ERK) antibodies showed that Ang II-activated EGF receptor and ERK. Both EGF receptor kinase inhibitor AG1478 and MEK1 inhibitor PD98059 attenuated ERK activation and DNA synthesis enhanced by Ang II. These results indicate that Ang II stimulates PSC proliferation through EGF receptor transactivation-ERK activation pathway.     

Angiotensin stimulates phosphatidylcholine synthesis via a pathway involving diacylglycerol, protein kinase C, ERK1/2, and CTP:phosphocholine cytidylyltransferase

We are probing the regulation of phosphatidylcholine (PC) synthesis by angiotensin II. In the accompanying paper, we showed that manipulation of the lipid second messengers, arachidonic acid or hydroxyeicosatetraenoic acid, produced downstream of the angiotensin AT1a receptor did not affect the PC synthesis rates in a manner consistent with direct activation of the rate limiting enzyme in the pathway, CTP:phosphocholine cytidylyltransferase (CCT). However, suppression of diacylglycerol (DAG) production with an inhibitor of phospholipase C-beta reduced angiotensin-dependent PC synthesis as well as ERK1/2 phosphorylation. Here, we show that the stimulation of PC synthesis and activation of CCT by angiotensin requires a signaling pathway that involves protein kinase C and ERK1/2. The inhibitors bis-indolylmaleimide I and PD98059 blocked ERK1/2 phosphorylation and completely eliminated angiotensin stimulation of the CCT-catalyzed reaction and PC synthesis. Exogenous addition of DAG using a lipid vesicle delivery system exactly mimicked the kinetics of angiotensin-promoted PC synthesis, suggesting that this mode of DAG delivery can effectively substitute for the DAG generated downstream of the activated AT1a receptor. Moreover, exogenous DAG activated ERK1/2, and the activation of PC synthesis by DAG was blocked by inhibition of protein kinase C and MEK. These data suggest that angiotensin-dependent DAG and the exogenously supplied DAG stimulate PC synthesis, not solely by direct action on CCT, but via a signaling pathway involving protein kinase C and ERK1/2. Angiotensin did not alter the net phosphorylation state of CCT as probed by immunoprecipitation of 32P-labeled CCT. Angiotensin stimulation of ERK1/2 likely mediates effects on CCT via a process other than CCT dephosphorylation.     

Angiotensin II stimulates rat astrocyte mitogen-activated protein kinase activity and growth through EGF and PDGF receptor transactivation

We showed that the intracellular tyrosine kinases src and pyk2 mediate angiotensin II (Ang II) stimulation of growth and ERK1/2 mitogen-activated protein (MAP) kinase phosphorylation in astrocytes. In this study, we investigated whether the membrane-bound receptor tyrosine kinases platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors mediate Ang II stimulation of ERK1/2 and astrocyte growth. Ang II significantly stimulated PDGF and EGF receptors in a dose- and time-dependent manner. The PDGF receptor and the EGF receptor were maximally stimulated with 100 nM Ang II (0.98+/-0.18- and 4.4+/-1.4-fold above basal, respectively). This stimulation occurred as early as 5 min, and was sustained for at least 15 min for both receptor tyrosine kinases. Moreover, 1 microM AG1478 and 0.25 microM PDGFRInhib attenuated Ang II stimulation of the EGF and PDGF receptors, respectively. Ang II-induced phosphorylation of ERK1/2 and astrocyte growth was mediated by both PDGF and EGF receptors. This report also provides novel findings that co-inhibiting EGF and PDGF receptors had a greater effect to decrease Ang II-induced ERK1/2 (90% versus 49% and 71% with PDGF receptor and EGF receptor inhibition, respectively), and astrocyte growth (60% versus 10% and 32% with PDGF receptor and EGF receptor inhibition, respectively). In conclusion we showed in astrocytes that the PDGF and the EGF receptors mediate Ang II-induced ERK1/2 phosphorylation and astrocyte growth and that these two receptors may exhibit synergism to regulate effects of the peptide in these cells.     

Angiotensin II stimulates phosphorylation of nuclear lamins via a protein kinase C-dependent mechanism in cultured vascular smooth muscle cells

Angiotensin II (ang II) induces c-fos gene expression in part via a protein kinase C-dependent mechanism in cultured vascular smooth muscle cells (VSMC). However, little is known about the mechanisms by which protein kinase C regulates nuclear functions. We examined the ability of ang II to phosphorylate nuclear lamina proteins in VSMC and the possibility that protein kinase C is involved in these putative phosphorylation events. Ang II stimulated the phosphorylation of Triton X-100- and high salt-insoluble nuclear envelope proteins with molecular weights of 70,000, 67,000, and 60,000. These proteins were identified as lamins A, B, and C, respectively, based on their mobilities on two-dimensional gel electrophoresis and interaction with antibodies to lamins as detected by immunoblot analyses. After a 2-min delay, phosphorylation levels of lamins increased, peaked at 20-30 min, and were sustained for at least 60 min after ang II stimulation. The threshold, half-maximal, and maximal concentrations of ang II which induced phosphorylation of lamins were 0.1, 0.5-1, and 100 nM, respectively. Phorbol 12-myristate 13-acetate also induced these reactions, whereas ionomycin did not. Down-regulation of protein kinase C by prolonged treatment with phorbol 12,13-dibutyrate attenuated ang II-induced phosphorylation of lamins. In vitro phosphorylation of nuclear envelope proteins by protein kinase C revealed that lamins served as substrates for this enzyme. These results indicate that ang II induces phosphorylation of lamins in cultured VSMC and suggest that protein kinase C is either directly or indirectly involved in these reactions. The results raise the possibility that phosphorylation of nuclear proteins is one of the important steps by which the protein kinase C signaling pathway regulates agonist-induced nuclear events.     

Sphingosine stimulates cellular proliferation via a protein kinase C-independent pathway

Sphingosine, a metabolite of membrane sphingolipids, is generally considered to be cytotoxic for a variety of cell types. However, we have found that sphingosine at low concentrations stimulates DNA synthesis and acts synergistically with known growth factors to induce proliferation of quiescent Swiss 3T3 fibroblasts. Structurally related analogs of sphingosine, such as N-stearoylsphingosine and other long chain aliphatic amines, had no mitogenic effects, suggesting that sphingosine did not induce nonspecific membrane perturbations. Sphingosine, which has been proposed to be a physiological inhibitor of protein kinase C, also markedly potentiates the mitogenic effect of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Sphingosine still stimulates DNA synthesis in cells made protein kinase C deficient by prolonged treatment with phorbol ester. At mitogenic concentrations, sphingosine does not bind to protein kinase C as shown by its lack of effect on phorbol dibutyrate binding. Only at higher concentrations, in the cytotoxic range, was there a displacement of phorbol dibutyrate from its cellular-binding sites. In contrast to sphingosine, H-7, a known inhibitor of protein kinase C, inhibited the mitogenic response to TPA and the TPA-induced phosphorylation of the 80 kDa cellular substrate of protein kinase C. Our results suggest that sphingosine may play an important role as a positive regulator of cell growth acting in a fundamentally different, protein kinase C-independent pathway.     

Angiotensin II stimulates vimentin phosphorylation via a Ca2+-dependent, protein kinase C-independent mechanism in cultured vascular smooth muscle cells

Intermediate filaments have been proposed, via phosphorylation by protein kinase C, to be involved in sustained contraction of smooth muscle. We examined the effect of angiotensin II on the phosphorylation of the intermediate filament protein, vimentin, in cultured rat aortic vascular smooth muscle cells. Angiotensin II induced phosphorylation of a Triton X-100- and high salt-insoluble protein with a molecular weight of 58,000. This protein was identified as vimentin based on its specific interaction with anti-vimentin antibody as detected by immunoblot analysis. Angiotensin II-induced phosphorylation of vimentin was time- and dose-dependent. Phosphorylation was detectable at 15 s, peaked at 2 min after angiotensin II stimulation, and gradually declined to a new plateau which was sustained for at least 30 min. The threshold, half-maximal and maximal concentrations of angiotensin II that stimulated vimentin phosphorylation were 0.01, 0.1, and 10 nM, respectively. The Ca2+ ionophore, ionomycin, stimulated vimentin phosphorylation to the same extent as angiotensin II, whereas the protein kinase C-activating phorbol ester, phorbol 12-myristate 13-acetate, had only marginal effects on this reaction. Pretreatment of the cells with [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid attenuated angiotensin II- and ionomycin-induced vimentin phosphorylation to the same extent. Down-regulation of protein kinase C induced by prolonged treatment of the cells with phorbol 12,13-dibutyrate did not inhibit angiotensin II-induced vimentin phosphorylation. These results indicate that angiotensin II stimulates vimentin phosphorylation via a Ca2+-dependent, protein kinase C-independent mechanism in vascular smooth muscle cells and suggest that cytoskeletal proteins are major targets for angiotensin II-induced phosphorylation events.     

Angiotensin II stimulates intercellular adhesion molecule-1 via an AT1 receptor/nuclear factor-kappaB pathway in brain microvascular endothelial cells

Microvascular changes in the brain are significant causes of cerebral edema and ischemia injury. A number of studies suggest that angiotensin (Ang) II may be involved in the initiation and regulation of processes occurring in brain ischemia. We recently reported that Ang II injures brain microvascular endothelial cells (BMEC) partially via stimulating intercellular adhesion molecule-1 (ICAM-1) expression. However, the signaling cascade leading to Ang II-induced ICAM-1 expression in BMEC was unclear. The present study tested the hypothesis that Ang II induces ICAM-1 expression via an AT1 receptor/nuclear factor-kappaB (NF-kappaB) pathway in BMEC. Ang II directly stimulated the expression of ICAM-1 mRNA and protein in primary cultured BMEC. Ang II treatment also resulted in the degradation of IkappaBalpha and increase of NF-kappaB p65 subunit in the nucleus as well as the DNA binding activity of nuclear NF-kappaB. These effects were abolished by pretreatment with the selective AT1 receptor antagonists, losartan and compound EXP-2528, or losartan plus the AT2 receptor antagonist PD123319, but not by PD123319 alone. Moreover, there were no significant differences between the losartan and losartan plus PD123319 groups. These findings indicate that Ang II-induced ICAM-1 upregulation in brain microvascular endothelial cells may be mediated via an AT1 receptor/NF-kappaB pathway.     

Angiotensin II stimulates renal proximal tubule Na-ATPase activity through the activation of protein kinase C

Recently, our group described an AT₁-mediated direct stimulatory effect of angiotensin II (Ang II) on the Na⁺-ATPase activity of proximal tubules basolateral membranes (BLM) [Am. J. Physiol. 248 (1985) F621]. Data in the present report suggest the participation of a protein kinase C (PKC) in the molecular mechanism of Ang II-mediated stimulation of the Na⁺-ATPase activity due to the following observations: (i) the stimulation of protein phosphorylation in BLM, induced by Ang II, is mimicked by the PKC activator TPA, and is completely reversed by the specific PKC inhibitor, calphostin C; (ii) the Na⁺-ATPase activity is stimulated by Ang II and TPA in the same magnitude, being these effects abolished by the use of the PKC inhibitors, calphostin C and sphingosine; (iii) the Na⁺-ATPase activity is activated by catalytic subunit of PKC (PKC-M), in a similar and nonadditive manner to Ang II; and (iv) Ang II stimulates the phosphorylation of MARCKS, a specific substrate for PKC.     

Endothelin-1 stimulates c-fos mRNA expression in C6 glioma cells via MAP kinase pathway.

Exposure of C6 glioma cells to endothelin-1 (ET-1) caused dose-dependent (10(-11) M to 10(-7) M) increments in intracellular calcium concentration ([Ca2+]i) and c-fos mRNA expression (4.5-fold) that were abolished by the endothelinA receptor antagonist, BQ610, and by inhibition of phospholipase C with U73122. ET-1 stimulated c-fos mRNA expression was also inhibited by protein kinase C inhibition (chelerythrine) and by the MAP kinase kinase inhibitor PD98059, but not by inhibitors of tyrosine kinases, protein kinase A type I or II, calmodulin kinase II, or calcium channel blockade. C6 cells treated with ET-1 demonstrated a significant increase in MAP kinase activity as evidenced by Western blotting. These results indicate a mechanism of long-term signaling by ET-1 involving an ET(A) receptor-mediated, phospholipase C(beta)-linked pathway that is dependent on protein kinase C and MAP kinase activation.     

T cell antigen receptor engagement stimulates c-raf phosphorylation and induces c-raf-associated kinase activity via a protein kinase C-dependent pathway

The c-raf kinase has been shown to be activated following stimulation of several tyrosine kinase growth factor receptors. We examined changes in c-raf following engagement of the T cell receptor for antigen (TCR), a stimulus which activates both a non-receptor tyrosine kinase and protein kinase C (PKC). We found that activation of the T-cell receptor on the T cell hybridoma 2B4 causes a rapid and stoichiometric hyperphosphorylation of c-raf and an increase in c-raf-associated kinase activity. Phosphoamino acid analysis showed that the phosphorylation was entirely on serine residues. High-resolution phosphopeptide mapping showed the appearance of a single major new phosphopeptide with TCR stimulation. That phosphopeptide was shown to comigrate with the major new phosphopeptide induced in response to phorbol ester. When cells were depleted of PKC by pretreatment with high concentrations of phorbol ester, TCR stimulation was no longer capable of inducing c-raf-associated kinase activity. To determine whether activation of the tyrosine kinase alone would activate c-raf, we examined the 2B4 variant cell line FL.8. In response to Thy-1 stimulation, these cells activate the tyrosine kinase but not protein kinase C due to a deficiency in TCR eta chain expression. We found that in contrast to Thy-1 stimulation of 2B4 cells, stimulation of FL.8 cells does not lead to the induction of c-raf-associated kinase activity, although phorbol ester activates the kinase to an equivalent degree in both cells. We conclude that T cell receptor activation of c-raf occurs via phosphorylation by the serine/threonine kinase PKC. Activation of c-raf through PKC represents a mechanism distinct from that reported for tyrosine kinase growth factor receptors.     

Angiotensin II promotes the proliferation of activated pancreatic stellate cells by Smad7 induction through a protein kinase C pathway

Activated pancreatic stellate cells (PSCs) play major roles in promoting pancreatic fibrosis. We previously reported that angiotensin II (Ang II) enhances activated PSC proliferation through EGF receptor transactivation. In the present study, we elucidated a novel intracellular mechanism by which Ang II stimulates cellular proliferation. TGF-beta1 inhibits activated PSC proliferation via a Smad3 and Smad4-dependent pathway in an autocrine manner. We demonstrated that Ang II inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4. Furthermore, Ang II rapidly induced inhibitory Smad7 mRNA expression. Adenovirus-mediated Smad7 overexpression inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4, and potentiated activated PSC proliferation. PKC inhibitor Go6983 blocked the induction of Smad7 mRNA expression by Ang II. In addition, 12-O-tetradecanoyl-phorbol 13-acetate, a PKC activator, increased Smad7 mRNA expression. These results suggest that Ang II enhances activated PSC proliferation by blocking autocrine TGF-beta1-mediated growth inhibition by inducing Smad7 expression via a PKC-dependent pathway.     

Calcitonin gene-related peptide stimulates intracellular cAMP via a protein kinase C-controlled mechanism in human ocular ciliary epithelial cells.

Calcitonin gene-related peptides I and II (CGRP I and II) were found to stimulate cAMP levels by approximately 4-6 fold in human nonpigmented ciliary epithelial cells with half-maximal effective concentrations of 20 x 10(-10) and 3 x 10(-10) M, respectively. Prior exposure of cells to 6 x 10(-7) M phorbol 12-myristate, 13-acetate for 15 min resulted in a 40-50% inhibition of CGRP II-dependent cAMP stimulation. Phorbol didecanoate and dioctanoylglycerol also effectively inhibited, whereas 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C, had no effect. Staurosporine, a protein kinase C inhibitor, blocked the inhibition of cAMP formation by phorbol esters. cAMP stimulation by forskolin or cholera toxin was not inhibited by phorbol esters, suggesting that neither a Gs protein nor adenylyl cyclase is the site of inhibition by protein kinase C. These data therefore suggest that CGRP receptors are required for inhibition of adenylate cyclase by protein kinase C.     

Angiotensin II induces diverse signal transduction pathways via both Gq and Gi proteins in liver epithelial cells

Angiotensin II stimulates a biphasic activation of Raf-1, MEK, and ERK in WB liver epithelial cells. The first peak of activity is rapid and transient and is followed by a sustained phase. Angiotensin II also causes a rapid activation of p21^ras in these cells. Moreover, two Src family kinases (Fyn and Yes) were activated by angiotensin II in a time- and concentration-dependent manner. Microinjection of antibodies against Fyn and Yes blocked angiotensin II-induced DNA synthesis and c-Fos expression in WB cells, indicating an obligatory involvement of these tyrosine kinases in the activation of the ERK cascade by angiotensin II. Finally, substantial reduction of the angiotensin II-stimulated activation of Fyn, Raf-1, ERK, and expression of c-Fos by pertussis toxin pretreatment argues that G proteins of the G_i family as well as the G_q family are involved in angiotensin II-mediated mitogenic pathways in WB cells. J. Cell. Biochem. 69:63–71, 1998. © 1998 Wiley-Liss, Inc.     

Angiotensin II can regulate gene expression by the AP-1 binding sequence via a protein kinase C-dependent pathway

An expression vector containing three copies of the AP-1 binding element (TRE) upstream of a thymidine kinase promotor which controlled the expression of the chloramphenicol acetyl transferase (CAT) gene was transiently transfected into vascular smooth muscle (VSM) cells and a human hepatocarcinoma cell line, Hep G2. Twelve hours of angiotensin (Ang) II exposure stimulated significantly CAT expression by 3.4 fold and 2.7 fold in Hep G2 and VSM cells, respectively. AngII had no effect on CAT expression of a control vector. This AngII-induced stimulation was attenuated significantly by an AngII receptor antagonist, Sar1 Ile8 AngII, and abolished completely by a PKC inhibitor, staurosporine. Our data suggest that the TRE plays a crucial role in AngII-induced gene expression that is mediated by PKC. We concluded that TRE is one of the AngII-responsive elements.     

Identification of two distinct pathways of protein kinase Calpha down-regulation in intestinal epithelial cells

Signal transduction pathways are controlled by desensitization mechanisms, which can affect receptors and/or downstream signal transducers. It has long been recognized that members of the protein kinase C (PKC) family of signal transduction molecules undergo down-regulation in response to activation. Previous reports have indicated that key steps in PKCalpha desensitization include caveolar internalization, priming site dephosphorylation, ubiquitination of the dephosphorylated protein, and degradation by the proteasome. In the current study, comparative analysis of PKCalpha processing induced by the PKC agonists phorbol 12-myristate 13-acetate and bryostatin 1 in IEC-18 rat intestinal epithelial cells demonstrates that: (a) at least two pathways of PKCalpha down-regulation can co-exist within cells, and (b) a single PKC agonist can activate both pathways at the same time. Using a combined biochemical and morphological approach, we identify a novel pathway of PKCalpha desensitization that involves ubiquitination of mature, fully phosphorylated activated enzyme at the plasma membrane and subsequent down-regulation by the proteasome. The phosphatase inhibitors okadaic acid and calyculin A accelerated PKCalpha down-regulation and inhibitors of vesicular trafficking did not prevent degradation of the protein, indicating that neither internalization nor priming site dephosphorylation are requisite intermediate steps in this ubiquitin/proteasome dependent pathway of PKCalpha down-regulation. Instead, caveolar trafficking and dephosphorylation are involved in a second, proteasome-independent mechanism of PKCalpha desensitization in this system. Our findings highlight subcellular distribution and phosphorylation state as critical determinants of PKCalpha desensitization pathways.     

The human 5-HT1A receptor expressed in HeLa cells stimulates sodium-dependent phosphate uptake via protein kinase C

Regulation of phosphate uptake was studied in HeLa cell lines after transfection with DNA encoding the human 5-HT1A receptor. Phosphate uptake was saturable and greater than 90% sodium-dependent, with Vmax approximately 30-35% without changing Km. Treatment with 5-HT or the 5-HT1A-specific agonist 8-OH-2-(di-n-propylamino)1,2,3,4-tetrahydronaphthalene increased Vmax approximately 40% without affecting Km. This effect was blocked by pretreatment with the 5-HT1 antagonists, methiothepine and spiperone, or pertussis toxin. Surprisingly, the stimulation was not secondary to an inhibition of adenylyl cyclase because 5-HT stimulated phosphate uptake approximately 20% in the presence of 1 mM 8-Br-cAMP. Rather, the primary pathway linked to the stimulation of phosphate uptake involved activation of protein kinase C because (i) 5-HT measurably activated protein kinase C in these cells, (ii) activators of protein kinase C (phorbol esters and diacylglycerol analogues) stimulated phosphate uptake in these cells (iii) the half-maximal doses for 5-HT-induced phosphatidylinositol hydrolysis and stimulation of phosphate uptake were virtually equivalent, and both effects were equally sensitive to pertussis toxin, and (iv) the stimulation was markedly attenuated in cells made deficient in protein kinase C. These results demonstrate that the stimulation of phosphatidylinositol hydrolysis by the 5-HT1A receptor can generate physiologically measurable effects on cellular transport and suggest that such accessory pathways may play a prominent role in signal transduction.     

Peroxynitrite activates mitogen-activated protein kinase (MAPK) via a MEK-independent pathway: a role for protein kinase C.

In this study we show that phosphorylation of extracellular signal-regulated kinase (ERK1/2; also known as p44/42MAPK) following peroxynitrite (ONOO(-)) exposure occurs via a MAPK kinase (MEK)-independent but PKC-dependent pathway in rat-1 fibroblasts. ONOO(-)-mediated ERK1/2 phosphorylation was not blocked by MEK inhibitors PD98059 and U0126. Furthermore, no increase in MEK phosphorylation was detected upon ONOO(-) treatment. Staurosporine was used to investigate whether protein kinase C (PKC) is involved. This was confirmed by down-regulation of PKC by phorbol-12,13-dibutyrate, which resulted in significant reduction of ERK1/2 phosphorylation by ONOO(-), implying that activation of ERK by ONOO(-) depends on activation of PKC. Indeed, PKCalpha and epsilon were activated upon ONOO(-) exposure. When cells were treated with ONOO(-) in a calcium-free buffer, no activation of PKCalpha was detected. Concomitantly, a reduction of ERK1/2 phosphorylation was observed suggesting that calcium was required for translocation of PKCalpha and ERK phosphorylation by ONOO(-). Indeed, ONOO(-) exposure resulted in increased cytosolic calcium, which depended on the presence of extracellular calcium. Finally, data using G?6976, an inhibitor of calcium-dependent PKC activation, implied that ONOO(-)-mediated ERK1/2 phosphorylation depends on activation of a calcium-dependent PKC.     

Angiotensin II stimulates renal proximal tubule Na(+)-ATPase activity through the activation of protein kinase C

Recently, our group described an AT(1)-mediated direct stimulatory effect of angiotensin II (Ang II) on the Na(+)-ATPase activity of proximal tubules basolateral membranes (BLM) [Am. J. Physiol. 248 (1985) F621]. Data in the present report suggest the participation of a protein kinase C (PKC) in the molecular mechanism of Ang II-mediated stimulation of the Na(+)-ATPase activity due to the following observations: (i) the stimulation of protein phosphorylation in BLM, induced by Ang II, is mimicked by the PKC activator TPA, and is completely reversed by the specific PKC inhibitor, calphostin C; (ii) the Na(+)-ATPase activity is stimulated by Ang II and TPA in the same magnitude, being these effects abolished by the use of the PKC inhibitors, calphostin C and sphingosine; (iii) the Na(+)-ATPase activity is activated by catalytic subunit of PKC (PKC-M), in a similar and nonadditive manner to Ang II; and (iv) Ang II stimulates the phosphorylation of MARCKS, a specific substrate for PKC.     

Mechanical pressure-induced phosphorylation of p38 mitogen-activated protein kinase in epithelial cells via Src and protein kinase C

The aim of this study was to determine the impact of lentiviral transduction on primary murine B cells. Studying B cell activities in vivo or using them for tolerance induction requires that the cells remain unaltered in their biological behavior except for expression of the transgene. As we show here, murine B cells can efficiently be transduced by lentiviral, VSV-G-pseudotyped vectors without the necessity of prior activation. Culture with LPS gave enhanced transduction efficiencies but led to the upregulation of CD86 and proliferation of the cells. Transduction of naive B cells by lentiviral vectors was dependent on multiplicity of infection and did not lead to a concomitant activation. Furthermore, the transduced cells could be used for studies in the NOD mouse system without altering the onset of diabetes. We conclude that lentiviral gene transfer into naive B cells is a powerful tool for manipulation of B cells for therapeutic applications.