Prostacyclin release by rat cardiac fibroblasts: inhibition of collagen expression

Cardiac fibroblasts, as the source of extracellular matrix for the left ventricle, subserve important functions to cardiac remodeling and fibrotic development following myocardial infarction or with pressure-overload cardiac hypertrophy. The fibroblast may be the target cell for angiotensin-converting enzyme inhibitors (ACEI) that are cardioprotective and reverse collagen deposition and remodeling but whose mechanisms of action remain controversial. Because we previously documented phenotypic differences between cardiac fibroblasts from the spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) left ventricle, the present study evaluated whether phenotypic differences also exist in the release of endogenous arachidonic acid metabolites or in the activation of phospholipase D, and the importance of observed differences to the formation of collagen and the mechanism of action of ACEI. The experimental design compared endogenous sources of arachidonic acid with exogenous prelabeling of cells. Angiotensin II stimulated greater arachidonic acid release than bradykinin, and WKY cells were more responsive than SHR. The major prostanoid formed by cardiac fibroblasts was prostaglandin I2 (PGI2), with more prostacyclin production by WKY cells than SHR cells both under nonstimulated conditions and in response to angiotensin II or bradykinin. Beraprost, a PGI2 analogue, was shown to decrease growth rate and DNA synthesis of fibroblasts and to inhibit mRNA expression for collagen types I and III, with SHR cells being less responsive to beraprost than WKY cells. These results potentially implicate eicosanoid metabolism, particularly PGI2, in collagen formation, fibrotic development, and cardiac remodeling, and they imply that the SHR genetic hypertension model may be predisposed to excess cardiac fibrosis.     

Collagen type I modulates the platelet-derived growth factor (PDGF) regulation of the growth and expression of beta1 integrins by rat mesangial cells

Mesangial cell (MC) proliferation and the deposition of collagen type I (collagen I) are the major pathological features in many types of glomerulonephritis (GN). Recent work suggested that beta-integrins play a critical role in the cell proliferation and extracellular matrix (ECM) remodeling observed in tissue repair after injury. To examine the involvement of beta-integrins in MC proliferation in association with the interaction of MCs with pathological collagen I, we investigated the effect of a prominent mitogen, platelet-derived growth factor-BB (PDGF-BB) on the growth and expression of beta-integrins by MCs cultured on plastic or in a three-dimensional collagen I gel. Immunoprecipitation using 35S-metabolic labeling, flow cytometry and a 3H-thymidine-uptake analysis demonstrated that PDGF-BB stimulated the cell mitogenicity and the expression of alpha5beta1 integrin (a fibronectin receptor), but not alpha1beta1 integrin (a collagen and laminin receptor) of MCs on plastic, in a dose-dependent manner. In contrast, MCs in the collagen I gels showed no significant changes in mitogenicity or alpha1beta1 and alpha5beta1 integrin expression, but increased alpha1beta1 integrin-mediated gel contraction was observed after PDGF-BB stimulation. Thus, the parallel up-regulation of MC-mitogenicity and alpha5beta1 integrin expression by PDGF-BB suggested that alpha5beta1 integrin is an important ECM receptor involved in the proliferative phenotype of MC. A spatial interaction between MCs and pathological collagen I in GN may influence the PDGF regulation of the MC phenotype regarding the cell growth and the expression of beta1 integrins.     

Effect of histamine on human fibroblast in vitro

The effects of histamine (CAS 51-45-6) on cell growth, collagen synthesis of fibroblasts derived from human foreskin, and on fibroblast-mediated collagen remodelling were studied. The cellmat DNA content was measured 2 days after human fibroblasts were plated at a split ratio of 1:10. Effect of histamine (10(-9)-10(-4) mol/l) on the increase of DNA content was not observed. Fibroblasts at confluence were cultured with histamine only, and with pyrilamine or cimetidine in addition to histamine for 2 h. Type I procollagen C-peptide in the medium was measured by enzyme immunoassay and was corrected by DNA content. Type I collagen synthesis was stimulated by histamine (10(-6)-10(-4) mol/l) and its stimulation was inhibited by cimetidine, but not by pyrilamine. Collagen solution containing fibroblast was incubated until gelation. It was incubated with histamine only, and with pyrilamine or cimetidine in addition to histamine. The gel contraction was stimulated by histamine (10(-4) mol/l) and its stimulation was inhibited by pyrilamine, but not by cimetidine. These facts suggests that histamine stimulates type I collagen synthesis of fibroblast and collagen remodeling via H2 and H1 receptors, respectively.     

Linking cell-fate specification to planar polarity: determination of the R3/R4 photoreceptors is a prerequisite for the interpretation of the Frizzled mediated polarity signal

Hypertensive cardiac hypertrophy is associated with the accumulation of collagen in the myocardial interstitium. Previous studies have demonstrated that this myocardial fibrosis accounts for impaired myocardial stiffness and ventricular dysfunction. Although cardiac fibroblasts are responsible for the synthesis of fibrillar collagen, the factors that regulate collagen synthesis in cardiac fibroblasts are not fully understood. We investigated the effects of angiotensin II on cardiac collagen synthesis in cardiac fibroblasts. Cardiac fibroblasts of 10 week old spontaneously hypertensive rats and age-matched Wistar-Kyoto rats were prepared and maintained in culture medium supplemented with 10% fetal calf serum. The expression of mRNA of the renin-angiotensin system (renin, angiotensinogen, angiotensin converting enzyme) was determined by using a ribonuclease protection assay. Basal collagen synthesis in cardiac fibroblasts from spontaneously hypertensive rats was 1.6 fold greater than that in the cell of Wistar-Kyoto rats. Angiotensin II stimulated collagen synthesis in cardiac fibroblasts in a dose-dependent manner. The responsiveness of collagen production to angiotensin II was significantly enhanced in cardiac fibroblasts from spontaneously hypertensive rats (100 nM angiotensin II resulted in 185 +/- 18% increase above basal levels, 185 +/- 18 versus 128 +/- 19% in Wistar-Kyoto rats p < 0.01). This effect was receptor-specific, because it was blocked by the competitive inhibitor saralasin and MK 954. These results indicate that collagen production was enhanced in cardiac fibroblasts from spontaneously hypertensive rats, that angiotensin II had a stimulatory effect on collagen synthesis in cardiac fibroblasts, and that cardiac fibroblasts from spontaneously hypertensive rats were hyper-responsive to stimulation by angiotensin II. Level of angiotensin and renin mRNA expressed in ventricles, and angiotensinogen mRNA expressed in fibroblasts from SHR were higher than those from WKY. These findings suggest that the cardiac renin-angiotensin system may play an important role in collagen accumulation in hypertensive cardiac hypertrophy.     

Pharmacologic regulation of Dupuytren's fibroblast contraction in vitro

Dupuytren's disease is associated with contraction of specialized fibroblasts present in the diseased palmar fascia. Pharmacologic agents were evaluated for their ability to promote or inhibit contraction of Dupuytren's fibroblasts in vitro using a collagen lattice contraction assay. In the first part of the study, lysophosphatidic acid (LPA), serotonin, angiotensin II, and prostaglandin F2 alpha were tested for their ability to promote Dupuytren's fibroblast contraction. Lysophosphatidic acid was found to significantly promote Dupuytren's fibroblast contraction as compared with controls. This response to LPA is dose dependent, with a half-maximal response of 0.07 microM. Angiotensin II, serotonin, and prostaglandin F2 alpha at 1 mM, induced a significant amount of contraction as compared to controls, but the amount of contraction was at least six times less than that observed for LPA. In the second part of the study, prostaglandins E1 and E2 or the calcium blockers nifedipine and verapamil were tested for their ability to inhibit LPA-promoted contraction. It was found that both types of inhibitors partially block LPA-promoted contraction of Dupuytren's fibroblasts. The effect of the various pharmacologic agents on normal palmar fibroblasts was not evaluated. The focus of this study was to examine the regulation of contraction of Dupuytren's fibroblasts. This study demonstrates that LPA is a potent agonist of Dupuytren's fibroblast contraction and that this contraction can be inhibited by specific pharmacologic agents. These findings provide a rational basis for investigating further the clinical use of the calcium channel blockers nifedipine or verapamil and prostaglandins E1 and E2 to control Dupuytren's disease and possibly other fibrotic conditions.     

Characterization of ovomucoid-specific T-cell lines and clones from egg-allergic subjects

Three-dimensional gel culture systems represent conditions that mimic the differentiated state of mesenchymal cells in vivo. We examined gel contraction, cell growth, and phenotypic modulation of rabbit arterial SMC in three-dimensional gel culture. The gel contraction rate was dependent on the collagen type; that is, the contraction by freshly isolated SMC was faster and more pronounced in type I collagen than in type III collagen. In contrast, the phenotypic modulation of SMC was independent of collagen type. The major portion of cells in both type I and III collagens with growth factors underwent transition from a contractile (G0 phase) to a synthetic phenotype (G1B phase), but this transition was clearly delayed compared with that on collagens. The cells had hardly begun DNA synthesis in either collagen type and failed to proliferate even after 10 days of culture. These results indicate that collagen type is important in gel contraction by vascular SMC, while the organization of collagen fibrils (two-dimensional vs three-dimensional) is more critical in the phenotypic transition and proliferation of these cells. However, the more specific organization of extracellular matrix than the collagen gel culture system may be necessary to maintain the contractile phenotype of SMC.     

Neonatal estrogen stimulates proliferation of periductal fibroblasts and alters the extracellular matrix composition in the rat prostate

The purpose of this study was to examine whether changes in extracellular matrix (ECM) molecules are associated with the growth inhibition and differentiation defects of the prostate gland following neonatal exposure to estradiol. Using immunocytochemistry (ICC), laminin and collagen IV were localized to the basement membrane (BM) as well to the basal lamina of the periductal smooth muscle of the control developing prostates. In contrast, fibronectin and collagen III were localized throughout the stromal ECM. Exposure to neonatal estrogen altered the staining profile for specific ECM molecules. In the estrogenized rats, a thick layer of cells negative for laminin and collagen IV was observed adjacent to the BM. Electron microscopy and ICC for alpha-actin, fibronectin, and vimentin identified this multicellular layer of periductal cells as differentiated fibroblasts. Peripheral to these fibroblasts, actin-positive smooth muscle formed a second layer of periductal stromal cells. PCNA labeling showed that estrogen exposure increased the fibroblast proliferation. Because many periductal fibroblasts were positive for estrogen receptor alpha (ER alpha) in estrogenized rats, a direct effect of estradiol on their proliferation is suggested. Gelatinolytic gels revealed that estrogen exposure did not alter the activity of matrix metalloproteinases associated with tissue remodeling during prostate morphogenesis. However, the periductal fibroblast layer in estrogenized prostates was devoid of urokinase- and tissue-plasminogen activator, which may potentially alter the localized proteolysis involved in matrix remodeling. It is proposed that proliferation of a multicellular layer of periductal fibroblasts in estrogenized prostates results in a physical barrier that constrains branching morphogenesis and blocks paracrine communications between smooth muscle and epithelial cells which normally regulate differentiation.     

Induction of collagenase-3 (MMP-13) expression in human skin fibroblasts by three-dimensional collagen is mediated by p38 mitogen-activated protein kinase

Collagenase-3 (matrix metalloproteinase-13, MMP-13) is a recently identified human MMP with an exceptionally wide substrate specificity and restricted tissue-specific expression. Here we show that MMP-13 expression is induced in normal human skin fibroblasts cultured within three-dimensional collagen gel resulting in production and proteolytic activation of MMP-13. Induction of MMP-13 mRNAs by collagen gel was potently inhibited by blocking antibodies against alpha1 and alpha2 integrin subunits and augmented by activating antibody against beta1 integrin subunit, indicating that both alpha1 beta1 and alpha2 beta1 integrins mediate the MMP-13-inducing cellular signal generated by three-dimensional collagen. Collagen-related induction of MMP-13 expression was dependent on tyrosine kinase activity, as it was abolished by treatment of fibroblasts with tyrosine kinase inhibitors genistein and herbimycin A. Contact of fibroblasts to three-dimensional collagen resulted in simultaneous activation of mitogen-activated protein kinases (MAPKs) in three distinct subgroups: extracellular signal-regulated kinase (ERK)1 and ERK2, Jun N-terminal kinase/stress-activated protein kinase, and p38. Induction of MMP-13 expression was inhibited by treatment of fibroblasts with a specific p38 inhibitor, SB 203580, whereas blocking the ERK1,2 pathway (Raf/MEK1,2/ERK1,2) by PD 98059, a selective inhibitor of MEK1,2 activation potently augmented MMP-13 expression. Furthermore, specific activation of ERK1,2 pathway by 12-O-tetradecanoylphorbol-13-acetate markedly suppressed MMP-13 expression in dermal fibroblasts in collagen gel. These results show that collagen-dependent induction of MMP-13 in dermal fibroblasts requires p38 activity, and is inhibited by activation of ERK1,2. Therefore, the balance between the activity of ERK1,2 and p38 MAPK pathways appears to be crucial in regulation of MMP-13 expression in dermal fibroblasts, suggesting that p38 MAPK may serve as a target for selective inhibition of collagen degradation, e.g. in chronic dermal ulcers.     

Analysis of tear-protein patterns as a diagnostic tool for the detection of dry eyes

Vasoactive peptides like thrombin, angiotensin II and endothelin induce vascular smooth muscle cell (VSMC) contraction via activation of G-protein-coupled receptors. Recent studies have shown that they also induce VSMC migration and proliferation, processes which are important in the remodelling of the vasculature during embryogenesis and in the response to vascular injury. G-protein-coupled receptor-mediated mitogenic signals appear to be transmitted via a number of intracellular mechanisms which include proto-oncogene gene expression, G-protein-mediated protein translocation and tyrosine phosphorylation of growth factor receptor proteins, and activation of autocrine growth factor pathways. The ability of vasoactive peptides to have an impact on signalling cascades mediated by growth factor tyrosine kinase receptors may be important in the pathogenesis of diseases in the vasculature.     

Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor

The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of growth factor stimulation, and recent evidence suggests that G protein-coupled receptors transactivate growth factor receptors to transmit mitogenic effects. In the present study, we report the involvement of epidermal growth factor receptor (EGF-R) in Ang II-induced extracellular signal-regulated kinase (ERK) activation, c-fos gene expression, and DNA synthesis in cardiac fibroblasts. Ang II induced a rapid tyrosine phosphorylation of EGF-R in association with phosphorylation of Shc protein and ERK activation. Specific inhibition of EGF-R function by either a dominant-negative EGF-R mutant or selective tyrphostin AG1478 completely abolished Ang II-induced ERK activation. Induction of c-fos gene expression and DNA synthesis were also abolished by the inhibition of EGF-R function. Calmodulin or tyrosine kinase inhibitors, but not protein kinase C (PKC) inhibitors or downregulation of PKC, completely abolished transactivation of EGF-R by Ang II or the Ca2+ ionophore A23187. Epidermal growth factor (EGF) activity in concentrated supernatant from Ang II-treated cells was not detected, and saturation of culture media with anti-EGF antibody did not affect the Ang II-induced transactivation of EGF-R. Conditioned media in which cells were incubated with Ang II could not induce phosphorylation of EGF-R on recipient cells. Platelet-derived growth factor-beta receptor was not phosphorylated on Ang II stimulation, and Ang II-induced c-jun gene expression was not affected by tyrphostin AG1478. Our results demonstrated that in cardiac fibroblasts Ang II-induced ERK activation and its mitogenic signals are dominantly mediated by EGF-R transactivated in a Ca2+/calmodulin-dependent manner and suggested that the effects of Ang II on cardiac fibroblasts should be interpreted in association with the signaling pathways regulating cellular proliferation and/or differentiation by growth factors.     

Remodeling of collagen matrix by human tumor cells requires activation and cell surface association of matrix metalloproteinase-2

We assessed the functional significance of tumor cell-associated matrix metalloproteinase (MMP)-2 in extracellular matrix remodeling compared with that of the soluble enzyme by evaluating the contraction of three-dimensional collagen lattices by human glioma U251.3 and fibrosarcoma HT-1080 cell lines. In this model, the constitutive synthesis and activation of the MMP-2 proenzyme were modulated by stable transfections of tumor cells with cDNA encoding membrane type 1-MMP (MT1-MMP). The efficiency of transfected cells in contracting collagen lattices was shown to be dependent on the MT1-MMP-mediated activation of MMP-2 accompanied by cell surface association of activated MMP-2, on the cell-matrix interactions controlled by collagen-specific integrins, and on the integrity of actin and microtubule cytoskeletons. Each one of these mechanisms was essential but was not sufficient by itself in accomplishing gel contraction by MT1-MMP-transfected cells. Both MMP-2 activation and gel contraction by transfected glioma cells were inhibited by tissue inhibitor of metalloproteinase (TIMP)-2 and the recombinant COOH-terminal domain of MMP-2. However, the kinetics and mechanisms of their inhibitory effects were different, because TIMP-2 and the COOH-terminal domain of MMP-2 preferentially inhibited the MT1-MMP-dependent and autocatalytic steps of MMP-2 activation, respectively. By contrast, TIMP-1, an efficient inhibitor of soluble MMP-2 activity, failed to affect gel contraction. In addition, soluble MMP-2 activated by either organomercurials or cells was not able to induce the contraction of collagen lattices when added to transfected cells. Therefore, soluble activated MMP-2, sensitive to TIMP-1 inhibition, does not mediate collagen gel contraction by tumor cells, whereas the activity of cell surface-associated MMP-2 plays a critical role in remodeling of the extracellular matrix in vitro. These mechanisms of functional and spatial regulation of MMP-2 may also be applicable to different aspects of tissue reorganization in vivo, including cell migration and invasion, angiogenesis, and wound healing.     

Report of the Eleventh International Symposium of the Foundation for Promotion of Cancer Research: Basic and Clinical Research in Gastric Cancer

Classically the AT1 receptors to angiotensin II are considered to be present on the smooth muscle cell membrane in the arterial wall, in which they diffusely regulate peripheral resistances. They are also present on numerous other cell types, including fibroblasts and myofibroblasts, monocytes and macrophages, endothelial cells, where they participate to the phenotypic modulation of the cell, involved in their activation leading to tissular remodeling. The intra-cellular pathway involving the phospholipase C activation and the mobilization of intra-cellular calcium is predominantly involved in the functional vasomotor response to angiotensin II. In contrast the intra-cellular signaling pathway leading to production of oxygen free radicals and activation of the NF-kappa-B system is probably mainly involved in the phenotypic modulation of target cells and their consequences on the vascular tissue remodeling.     

The role of self-treatment guidelines in self-management education for adult asthmatics

In cardiac fibroblasts, angiotensin II (Ang II) induced a rapid increase in extracellular signal regulated kinase (ERK) activity in a pertussis toxin insensitive manner. This ERK activation was abolished by the Gq-associated phospholipase C inhibitor U73122 but was insensitive to protein kinase C (PKC) inhibitors or PKC downregulation by phorbol ester. Intracellular Ca2+ chelation by BAPTA-AM or TMB-8 abolished Ang II induced ERK activation, whereas treatment with EGTA or nifedipine did not affect it. Ca2+ ionophore A23187 also induced a rapid increase in ERK activity to an extent similar to that of Ang II stimulation. Calmodulin inhibitors (W7 and calmidazolium) and tyrosine kinase inhibitors (genistein and ST638) completely blocked ERK activation by Ang II and A23187. Both Ang II and A23187 caused a rapid increase in the binding of GTP to p21(Ras), which was nearly abolished by genistein and calmidazolium. Transfection with the dominant negative mutant of Ras and the Ras inhibitor manumycin completely inhibited Ang II induced ERK activation. It was also found for the first time that cardiac fibroblasts abundantly expressed Ca2+-sensitive tyrosine kinase Pyk2/CAKbeta/RAFTK and that Ang II markedly induced its activation in a Ca2+/calmodulin-sensitive manner. Overexpression of the dominant negative mutant of Pyk2 significantly attenuated Ang II or A23187-induced ERK activities (36% and 38% inhibition compared with that in mock-transfected cells, respectively) and ERK tyrosine phosphorylation levels, as well as an increase in the binding of GTP to p21(Ras). These findings demonstrate that in cardiac fibroblasts, Ang II induced Ras/ERK activation is dominantly regulated by Gq-coupled Ca2+/calmodulin signaling and that Pyk2 plays an important role in the signal transmission for efficient activation of the Ang II induced Ras/ERK pathway.     

Angiotensin II-induced association of phospholipase Cgamma1 with the G-protein-coupled AT1 receptor

An early event in signaling by the G-protein-coupled angiotensin II (Ang II) AT1 receptor in vascular smooth muscle cells is the tyrosine phosphorylation and activation of phospholipase Cgamma1 (PLCgamma1). In the present study, we show that stimulation of this event by Ang II in vascular smooth muscle cells is accompanied by binding of PLCgamma1 to the AT1 receptor in an Ang II- and tyrosine phophorylation-dependent manner. The PLCgamma1-AT1 receptor interaction appears to depend on phosphorylation of tyrosine 319 in a YIPP motif in the C-terminal intracellular domain of the AT1 receptor and binding of the phosphorylated receptor by the most C-terminal of two Src homology 2 domains in PLCgamma1. PLCgamma1 thus binds to the same site in the receptor previously identified for binding by the SHP-2 phosphotyrosine phosphatase.JAK2 tyrosine kinase complex. A single site in the C-terminal tail of the AT1 receptor can, therefore, be bound in a ligand-dependent manner by two different downstream effector proteins. These data demonstrate that G-protein-coupled receptors can physically associate with intracellular proteins other than G proteins, creating membrane-delimited signal transduction complexes similar to those observed for classic growth factor receptors.     

Growth hormone regulation of SIRP and SHP-2 tyrosyl phosphorylation and association

SIRPs (signal-regulatory proteins) are a family of transmembrane glycoproteins that were identified by their association with the Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2 in response to insulin. Here we examine whether SIRPalpha and SHP-2 are signaling molecules for the receptors for growth hormone (GH), leukemia inhibitory factor (LIF), or interferon-gamma (IFNgamma), cytokine receptor superfamily members that bind to and activate Janus kinase 2 (JAK2). In 3T3-F442A fibroblasts, GH rapidly stimulates tyrosyl phosphorylation of both SIRPalpha and SHP-2 and enhances association of SHP-2 with SIRPalpha. Consistent with JAK2 binding and phosphorylating SIRPalpha in response to GH, co-expression of SIRPalpha and JAK2 in COS cells results in tyrosyl phosphorylation of SIRPalpha and JAK2 association with SIRPalpha. LIF does not stimulate tyrosyl phosphorylation of SIRPalpha but stimulates greater tyrosyl phosphorylation of SHP-2 than GH. Additionally, LIF enhances association of SHP-2 with the gp130 subunit of the LIF receptor signaling complex. IFNgamma, which stimulates JAK2 to a greater extent than LIF, is ineffective at stimulating tyrosyl phosphorylation of SIRPalpha or SHP-2. These results suggest that SIRPalpha is a signaling molecule for GH but not for LIF or IFNgamma. Differential phosphorylation of SIRPalpha and SHP-2 may contribute to the distinct physiological effects of these ligands.     

Regulation of radiation-induced apoptosis in oncogene-transfected fibroblasts: influence of H-ras on the G2 delay

The high-affinity receptor (R) for IL-5 consists of a unique alpha chain (IL-5R alpha) and a beta chain (beta c) that is shared with the receptors for IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF). We defined two regions of IL-5R alpha for the IL-5-induced proliferative response, the expression of nuclear proto-oncogenes, and the tyrosine phosphorylation of cellular proteins including beta c, SH2/SH3-containing proteins and JAK2 kinase. In the studies described here, we demonstrate that IL-5, IL-3 or GM-CSF stimulation induces the tyrosine phosphorylation of JAK2, and to a lesser extent JAK1, and of STAT5. Mutational analysis revealed that one of the proline residues, particularly Pro352 and Pro355, in the membrane-proximal proline-rich sequence (Pro352-Pro353-X-Pro355) of the cytoplasmic domain of IL-5R alpha is required for cell proliferation, and for both JAK1 and JAK2 activation. In addition, transfectants expressing chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c responded to IL-5 for proliferation and tyrosine phosphorylation of JAK1. Intriguingly, electrophoretic mobility shift assay analysis revealed that STAT5 was activated in cells showing either JAK1 or JAK2 tyrosine phosphorylation. These results indicate that activation of JAK1, JAK2 and STAT5 is critical to coupling IL-5-induced tyrosine phosphorylation and ultimately mitogenesis, and that Pro352 and Pro355 in the proline-rich sequence appear to play more essential roles in cell growth and in both JAK1/STAT5 and JAK2/STAT5 activation than Pro353 does.     

Epidermal growth factor and angiotensin II regulation of extracellular signal-regulated protein kinase in rat liver epithelial WB cells

Activation of extracellular signal-regulated protein kinase (ERK) is considered essential for mitogenesis. In the present study, rat liver epithelial WB cells were used to investigate the relative roles of Ca2+, protein kinase C (PKC), and protein tyrosine phosphorylation in mitogenesis and activation of the ERK pathway stimulated by epidermal growth factor (EGF) and angiotensin II (Ang II). The sensitivity of the ERK pathway to Ca2+ was studied by using 1,2-bis (O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) to chelate intracellular Ca2+ and a low extracellular Ca2+ concentration to prevent Ca2+ influx. Agonist-induced PKC activation was diminished by inhibition of PKC by GF-109203X (bisindolylmaleimide) or by down-regulation of PKC by long-term treatment of the cells with phorbol myristate acetate (PMA). Our results show that although activation of PKC was critical for mitogenesis induced by Ang II or EGF, the initial activation of ERK by both agonists in these cells was essentially independent of PKC activation and was insensitive to Ca2+ mobilization. This is in contrast to the findings in some cell types that exhibit a marked dependency on mobilization of Ca2+ and/or PKC activation. On the other hand, an obligatory tyrosine phosphorylation step for activation of ERK was indicated by the use of protein tyrosine kinase inhibitors, which profoundly inhibited the activation of ERK by EGF, Ang II, and PMA. Additional experiments indicated that tyrosine phosphorylation by a cytosolic tyrosine kinase may represent a general mechanism for G-protein coupled receptor mediated ERK activation.