Focal adhesion kinase promotes phospholipase C-γ1 activity

The nonreceptor tyrosine kinase FAK ("focal adhesion kinase") is a key mediator of integrin signaling events controlling cellular responses to the extracellular matrix, including spreading, migration, proliferation, and survival. Integrin-ligand interactions stimulate FAK tyrosine phosphorylation and activation of FAK signaling functions. Here evidence is presented that the FAK autophosphorylation site Tyr-397 mediates a direct interaction with the C-terminal Src homology 2 domain of phospholipase C (PLC)-gamma1 and that this is required for both adhesion-dependent association of the two molecules and increased inositol phosphate production in mouse embryo fibroblasts. Overexpression of FAK and PLC-gamma1 in COS-7 cells increases PLC-gamma1 enzymatic activity and tyrosine phosphorylation, also dependent on FAK Tyr-397. However, FAK appears incapable of directly phosphorylating PLC-gamma1. These observations suggest a role for FAK in recruiting PLC-gamma1 to the plasma membrane at sites of cell-matrix adhesion and there promoting its enzymatic activity, possibly by releasing the repression caused by intramolecular interactions of the PLC-gamma1 Src homology domains and/or by positioning it for phosphorylation by associated Src-family kinases. These findings expand the known signaling functions of FAK and provide mechanistic insight into integrin-stimulation of PLC-gamma1.     

Involvement of PI3K and ERK1/2 pathways in hepatocyte growth factor-induced cholangiocarcinoma cell invasion

AIM:To investigate the role of hepatocyte growth factor(HGF) in cholangiocarcinoma(CCA) cell invasiveness and the mechanisms underlying such cellular responses. METHODS:Effects of HGF on cell invasion and motility were investigated in two human CCA cell lines,HuCCA-1 and KKU-M213,using Transwell in vitro assay.Levels of proteins of interest and their phosphorylated forms were determined by Western blotting.Localization of E-cadherin was analyzed by immunofluorescence staining and visualized under confocal m...     

Mechanisms of hepatocyte growth factor-mediated vascular smooth muscle cell migration

The migration of vascular smooth muscle cells (SMCs) from the media into the neointima and their subsequent proliferation is important in the pathogenesis of atherosclerosis. This process is regulated by multiple factors, including growth factors, and involves changes in the interaction of SMCs with the extracellular matrix and in intracellular signaling cascades that regulate cell movement. We demonstrated previously that hepatocyte growth factor (HGF) is expressed in human atherosclerotic plaques. Although HGF has been shown to promote SMC migration, the mechanisms involved in this process have not been characterized fully. In this study, inhibitory antibodies were used to determine which integrins mediated HGF-induced SMC migration. Inhibition of beta1 or beta3 integrin resulted in a significant decrease in migration. Subsequent experiments were performed to characterize additional biochemical mechanisms involved in HGF-mediated migration. HGF induced the redistribution of focal adhesions, the activation of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) and their increased association with beta1 and beta3 integrins, and the production of pro-matrix metalloproteinase-2. Migration levels were significantly reduced by cotreatment of SMCs with the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor, UO126, the p38 inhibitor, SB203580, or the phosphatidylinositol-3 kinase inhibitor, LY294002. In HGF-treated SMCs, focal adhesion redistribution and FAK and Pyk2 activation were decreased by ERK1/2 inhibition. Neither SB203580 nor LY294002 inhibited HGF-induced ERK1/2 activation. Thus, ERK1/2 signaling may play an important role in HGF-mediated SMC migration by contributing to focal adhesion redistribution and FAK and Pyk2 activation.     

Effect of Src kinase inhibition on metastasis and tumor angiogenesis in human pancreatic cancer

Tumor angiogenesis is a process that requires migration, proliferation, and differentiation of endothelial cells. We hypothesized that decrease in pancreatic tumor growth due to inhibition of Src activity is associated with the inability of Src kinase to trigger a network of such signaling processes, which finally leads to endothelial cell death and angiogenesis-restricted tumor dormancy. The therapeutic efficacy of Src kinase inhibitor AZM475271 was tested in nude mice orthotopically xenografted with L3.6pl pancreatic carcinoma cells. No liver metastases and peritoneal carcinosis were detected and a significant effect on the average pancreatic tumor burden was observed following treatment with AZM475271, which in turn correlated with a decrease in cell proliferation and an increase in apoptotic endothelial cells. AZM475271 was shown to significantly inhibit migration of human umbilical vein endothelial cells in an in vitro Boyden Chamber cell migration assay. In a rat aortic ring assay we could demonstrate as well inhibition of endothelial cell migration and sprouting following therapy with Src kinase inhibitor at similar doses. The most conclusive anti-angiogenic activity of AZM475271 was demonstrated in vivo (mouse corneal micropocket assay) by showing a marked inhibition of basic fibroblast growth factor-induced neovascularization in response to systemic administration of AZM475271. Furthermore, we could show reduced proliferation of HUVECs determined with the TACS MTT Cell Viability Assay Kit. The blockade of Src kinase significantly reduced the level of VEGF in L3.6pl medium, the effect which was found also in the cell culture supernate from HUVECs. Inhibition of Src kinase by AZM475271 also showed prevention of survival signaling from VEGF and EGF receptors. Treatment with AZM475271 resulted in VEGF - dependent inhibition of tyrosine phosphorylation of FAK. HUVECs were also examined using propidium iodide staining for cell cycle analysis by FACS. Inhibition of Src kinase promoted HUVEC apoptosis in a dose-dependent manner. Taken together, our results suggest that the Src kinase inhibitor AZM475271, in addition to its effects on tumor cells, suppresses tumor growth and metastasis in vitro and in vivo potentially also by anti-angiogenic mechanisms.     

Src及其抑制剂对非小细胞肺癌作用的研究进展

Src酪氨酸激酶家族是非小细胞肺癌（NSCLC）细胞生存、增生、游走和黏附的关键调控点。Src可被黏着斑激酶（FAK）、表皮生长因子（EGF）、肝细胞生长因子（HGF）、血小板衍生生长因子（PDGF）等激活并参与多条信号转导途径。Src的过度激活在NSCLC的发生、发展和转移过程中起着非常重要的作用。Src抑制剂-Dasatinib、AZD0530、SKI-606、XL999和M475271的体外实验和临床应用有抑制NSCLC的作用。     

Involvement of 85-kd cytosolic phospholipase A(2) and cyclooxygenase-2 in the proliferation of human cholangiocarcinoma cells

Cyclooxygenase 2 (COX-2) and cytosolic phospholipase A(2) (cPLA(2)) are the crucial rate-limiting enzymes in prostaglandin (PG) metabolism that show increased expression in a number of human cancers, including cholangiocarcinomas; and treatment of cholangiocarcinoma cell lines with COX-2 inhibitors can decrease proliferation. Cholangiocarcinomas also produce and proliferate in response to nonneoplastic biliary epithelial cell mitogens, such as interleukin 6 (IL-6) and hepatocyte growth factor (HGF). This study was designed to determine whether there is any relationship between eicosanoid metabolism and growth stimulation by IL-6 and HGF, two important biliary epithelial cell and cholangiocarcinoma mitogens. Incubation of SG231, a well-characterized human cholangiocarcinoma cell line, with HGF, IL-6, PGE(2), or PGF(2)alpha resulted in significantly increased cell growth. HGF and IL-6 also induced a rapid release of arachidonic acid (AA) from SG231 and increased the synthesis of PGE(2) and PGF(2)alpha. The cPLA(2) inhibitor arachidonyl fluorophosphonate (MAFP) and the COX-2 inhibitor NS-398 significantly inhibited HGF- and IL-6-induced release of AA, PG synthesis, and proliferation in SG231 cells as well as two other human cholangiocarcinoma cell lines, HuCCT1 and CC-LP-1 cells. Thus, PGs alone can induce cholangiocarcinoma growth, and the HGF- and IL-6-induced proliferation is mediated, at least in part, by PGs. HGF and IL-6 also induced a rapid phosphorylation of cPLA(2) (within 1 minute) but did not alter cPLA(2) and COX-2 protein expression. The HGF- and IL-6-induced cPLA(2) phosphorylation was blocked by the inhibitors of p38 and p42/44 MAP kinases, protein kinase C, calmodulin kinase, and tyrosine kinase, showing that HGF- and IL-6-induced AA release and PG production are mediated by phosphorylation of cPLA(2). In conclusion, molecular pathways link classic biliary epithelial cell mitogens to PG metabolism constituents in cholangiocarcinoma growth, which may be exploited as potential therapeutic targets.     

Proline-rich tyrosine kinase 2 and Src kinase signaling transduce monosodium urate crystal-induced nitric oxide production and matrix metalloproteinase 3 expression in chondrocytes

OBJECTIVE: Articular deposition of monosodium urate monohydrate (MSU) crystals may promote cartilage and bone erosion. Therefore, the aim of this study was to determine how MSU crystals stimulate chondrocytes. METHODS: Nitric oxide (NO) release, and expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase 3 (MMP-3) were assessed in cultured chondrocytes treated with MSU. MSU-induced functional signaling by specific protein kinases (p38, Src, and the focal adhesion kinase [FAK] family members proline-rich tyrosine kinase 2 [Pyk-2] and FAK) was also examined using selective pharmacologic inhibitors and transfection of kinase mutants. RESULTS: MSU induced MMP-3 and iNOS expression and NO release in chondrocytes in a p38-dependent manner that did not require interleukin-1 (IL-1), as demonstrated by using IL-1 receptor antagonist. MSU induced rapid tyrosine phosphorylation of Pyk-2 and FAK, their adaptor protein paxillin, and interacting kinase c-Src. Pyk-2 and c-Src signaling both mediated p38 MAPK activation in response to MSU. Pyk-2 and c-Src signaling played a major role in transducing MSU-induced NO production and MMP-3 expression. But, despite the observed FAK phosphorylation, a selective pharmacologic FAK inhibitor and a FAK dominant-negative mutant both failed to block MSU-induced NO release or MMP-3 expression in parallel experiments. CONCLUSION: In chondrocytes, MSU crystals activate a signaling kinase cascade typically employed by adhesion receptors that involves upstream Src and FAK family activation and downstream p38 activation. In this cascade, Pyk-2, Src, and p38 kinases transduce MSU-induced NO production and MMP-3 expression. Our results identify Pyk-2 and c-Src as novel sites for potential therapeutic intervention in cartilage degradation in chronic gout.     

Roles of the MEK1/2 and AKT pathways in CXCL12/CXCR4 induced cholangiocarcinoma cell invasion

AIM:To evaluate the expression of C-X-C motif chemokine receptor 4(CXCR4)and its signaling cascades,which were previously identified as a key factor for cancer cell progression and metastasis,in cholangiocarcinoma cell lines.METHODS:The expression of CXCR4 and its signaling cascades were determined in the cholangiocarcinoma cell lines(RMCCA1 and KKU100)by Western blotting.The invasion assays and the detection of actin polymerization were tested in these cholangiocarcinoma cells treated with CXC chemokine ligand-12(CXCL12).RESULTS:Expression of CXCR4 was detected in both cholangiocarcinoma cell lines and activation of CXCR4 with CXCL12 triggered the signaling via the extracellular signal-regulated kinase-1/2(ERK1/2)and phosphoinositide 3-kinase(PI3K)and induction of cholangiocarcinoma cell invasion,and displayed high levels of actin polymerization.Addition of CXCR4 inhibitor(AMD3100)abrogated CXCL12-induced phosphorylation of MEK1/2 and Akt in these cells.Moreover,treatment with MEK1/2 inhibitor(U0126)or PI3K inhibitor(LY294 002)also attenuated the effect of CXCL12-induced cholangiocarcinoma cell invasion.CONCLUSION:These results indicated that the activation of CXCR4 and its signaling pathways(MEK1/2 and Akt)are essential for CXCL12-induced cholangiocarcinoma cell invasion.This rises Implications on a potential role for the inhibition of CXCR4 or its signal cascades in the treatment of cholangiocarcinoma.     

Involvement of integrins and Src in tauroursodeoxycholate-induced and swelling-induced choleresis

BACKGROUND & AIMS: Stimulation of canalicular secretion by tauroursodeoxycholate (TUDC) involves dual activation of p38 mitogen-activated protein kinase (p38(MAPK)) and extracellular signal-regulated kinase (ERK). This study investigates the sensing and upstream signaling events of TUDC-induced choleresis. METHODS: TUDC and hypo-osmolarity effects on protein kinase activities and taurocholate excretion were studied in perfused rat liver. RESULTS: TUDC induced a rapid activation of focal adhesion kinase (FAK) and Src, as shown by an increase in Y418 phosphorylation and a decrease in Y529 phosphorylation of Src. Inhibition of Src by PP-2 abolished the TUDC-induced activation of p38(MAPK) but not of FAK and ERKs. An integrin-inhibitory peptide with an RGD motif blocked TUDC-induced FAK, Src, ERK, and p38(MAPK) activation, suggesting that integrin signaling toward FAK/Src is required for TUDC-induced MAPK activation. The RGD peptide and PP-2 also abolished the stimulation of taurocholate excretion in perfused rat liver in response to TUDC. Integrin-dependent Src activation was also identified as an upstream event in hypo-osmotic signaling toward MAPKs and choleresis. CONCLUSIONS: TUDC-induced stimulation of canalicular taurocholate excretion involves integrin sensing, FAK, and Src activation as upstream events for dual MAPK activation. Integrins may also represent one long-searched sensor for cell hydration changes in response to hypo-osmolarity.     

Src family tyrosine kinases are activated by Flt3 and are involved in the proliferative effects of leukemia-associated Flt3 mutations

OBJECTIVE: The hematopoietic growth factor receptor, Fms-like tyrosine kinase-3 (Flt3), modulates survival and proliferation of myeloid and B-cell precursors. Activating mutations of Flt3 are the most common molecular abnormalities in acute myeloid leukemia (AML) and have an apparent role in leukemogenesis. However, signaling pathways mediating Flt3 effects are incompletely understood. The role of Src kinases is unknown, although some, such as Lyn, have also been linked to leukemogenesis. This study examines the role of Src kinases in Flt3 signaling and the oncogenic effects of leukemia-associated Flt3 mutations. MATERIALS AND METHODS: We examined the activation and functional roles of Src kinases in human leukemic myeloid cell lines expressing wild-type Flt3 or a constitutively active mutant, and in cells stably transduced with human wild-type or mutant Flt3. RESULTS: Flt3 ligand stimulation of wild-type Flt3 increased phosphorylation of Src kinase Lyn. Constitutive Lyn phosphorylation and activation was found in cells expressing constitutively active Flt3 mutants. Src kinases are implicated in downregulation of closely related receptors, but Src inhibitors had no effect on ligand-stimulated Flt3 degradation, or on the rapid degradation of an Flt3 mutant. However, growth-factor-independent proliferation resulting from mutant Flt3 expression did depend on the activity of Src kinases. CONCLUSION: Our studies reveal for the first time the involvement of Src kinases in Flt3 signaling, with activation of Lyn by constitutively active Flt3 mutants as well as ligand-stimulated wild-type receptor, and show that Src kinase inhibitors block proliferative effects of Flt3 mutants found in AML. Thus, Src kinases may represent targets for inhibitor therapy in Flt3-related AML.     

Imatinib mesylate induces apoptosis in human cholangiocarcinoma cells.

BACKGROUND: Cholangiocarcinoma is a highly malignant, usually fatal cancer with limited therapeutic options. Receptor tyrosine kinases contribute to the development and progression of this cancer. The relatively selective tyrosine kinase inhibitor imatinib mesylate (STI-571 or Gleevec(R)) has recently been licensed. However, the ability of this drug to inhibit signal transduction and induce apoptosis in human cholangiocarcinoma cells is incompletely studied. Thus, our goal was to examine the ability of STI-571 to induce apoptosis in KMCH-1 cells, a human cholangiocarcinoma cell line. METHODS: Apoptosis was assessed morphologically and also biochemically by measuring caspase activity and the mitochondrial membrane potential. STI-571 induced apoptosis and inhibited growth of KMCH-1 cells in a time- and concentration-dependent manner. The induction of apoptosis was accompanied by mitochondrial depolarization followed by a 4.5-fold increase in caspase activation and was abrogated by the pancaspase inhibitor z-VAD(OMe)-fmk. Interestingly, cholangiocarcinoma cells do not express detectable PDGFR, c-Abl or c-Kit, which are protein kinases known to be directly inhibited by STI-571. However, a significant decrease in epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) phosphorylation was observed following treatment with STI-571. This decrease in EGFR and FAK phosphorylation was associated with a reduction in Akt activity resulting in loss of Mcl-1, a potent anti-apoptotic Bcl-2 family protein. CONCLUSIONS: These results indicate that STI-571 induces caspase-dependent apoptosis in a human cholangiocarcinoma cell line and suggest that STI-571 might warrant further investigation as a possible agent for treatment of human cholangiocarcinoma.     

Pressure activates colon cancer cell adhesion by inside-out focal adhesion complex and actin cytoskeletal signaling

BACKGROUND AND AIMS: Few circulating tumor cells implant or cause metastasis. We hypothesized that venous or lymphatic pressure or iatrogenic pressure during resection activates signals governing malignant colonocyte adhesion. METHODS: We studied the effect of 15 mm Hg increased pressure for 30 minutes on adhesion of primary human colon cancer cells and SW620 colonocytes to collagen and endothelial cells. We modulated integrin affinity with extracellular cations. We assessed binding affinity by detachment assay; integrin surface expression by flow cytometry; and focal adhesion kinase (FAK), Src, and extracellular signal-regulated kinase (ERK) activation by Western analysis and Src in vitro kinase assay. We inhibited Src (PP2), FAK (small RNA interference, SiRNA, or FRNK transfection), MEK (PD98059), PKC (calphostin C), and actin destabilization (phalloidin). RESULTS: Pressure and manganese stimulated primary and SW620 colonocyte adhesion to collagen. Pressure also stimulated SW620 adhesion to endothelial monolayers. Pressure strengthened SW620 binding force to matrix without changing integrin surface expression. Pressure activated SW620 FAK and Src, but not ERK. Manganese did not. Calcium-inhibited adhesion but stimulated FAK (but not Src). PP2 prevented pressure activation of Src, Src phosphorylation of FAK576, and pressure-stimulated adhesion but not FAK397 autophosphorylation. FRNK transfection or FAK SiRNA also prevented pressure-stimulated adhesion. FAK SiRNA ablated pressure-activated FAK397, Src, and FAK576 phosphorylation. Neither Src nor FAK inhibition blocked cation effects. Phalloidin prevented pressure-stimulated adhesion. PD98059 or calphostin C did not. CONCLUSIONS: In contrast to divalent cations, extracellular pressure may increase integrin affinity and promote colon cancer adhesion via actin dependent inside-out FAK and Src signals. This mechanotransduced pathway may regulate metastasizing tumor cell adhesion.     

Proline‐rich tyrosine kinase 2 and Src kinase signaling transduce monosodium urate crystal–induced nitric oxide production and matrix metalloproteinase 3 expression in chondrocytes

Objective

Articular deposition of monosodium urate monohydrate (MSU) crystals may promote cartilage and bone erosion. Therefore, the aim of this study was to determine how MSU crystals stimulate chondrocytes.

Methods

Nitric oxide (NO) release, and expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase 3 (MMP‐3) were assessed in cultured chondrocytes treated with MSU. MSU‐induced functional signaling by specific protein kinases (p38, Src, and the focal adhesion kinase [FAK] family members proline‐rich tyrosine kinase 2 [Pyk‐2] and FAK) was also examined using selective pharmacologic inhibitors and transfection of kinase mutants.

Results

MSU induced MMP‐3 and iNOS expression and NO release in chondrocytes in a p38‐dependent manner that did not require interleukin‐1 (IL‐1), as demonstrated by using IL‐1 receptor antagonist. MSU induced rapid tyrosine phosphorylation of Pyk‐2 and FAK, their adaptor protein paxillin, and interacting kinase c‐Src. Pyk‐2 and c‐Src signaling both mediated p38 MAPK activation in response to MSU. Pyk‐2 and c‐Src signaling played a major role in transducing MSU‐induced NO production and MMP‐3 expression. But, despite the observed FAK phosphorylation, a selective pharmacologic FAK inhibitor and a FAK dominant‐negative mutant both failed to block MSU‐induced NO release or MMP‐3 expression in parallel experiments.

Conclusion

In chondrocytes, MSU crystals activate a signaling kinase cascade typically employed by adhesion receptors that involves upstream Src and FAK family activation and downstream p38 activation. In this cascade, Pyk‐2, Src, and p38 kinases transduce MSU‐induced NO production and MMP‐3 expression. Our results identify Pyk‐2 and c‐Src as novel sites for potential therapeutic intervention in cartilage degradation in chronic gout.

     

Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase.

The focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To gain insight into FAK function, we examined the potential interaction of FAK with intracellular signaling molecules containing the Src homology 2 domains. We report here the stable association of FAK with phosphatidylinositol 3-kinase (PI3-kinase; EC 2.7.1.137) in NIH 3T3 mouse fibroblasts. This interaction was stimulated by cell adhesion concomitant with FAK activation. We also found that recombinant FAK bound to the p85 subunit of PI 3-kinase directly in vitro and that autophosphorylation of recombinant FAK in vitro increased its binding to PI 3-kinase. We detected increased tyrosine phosphorylation of the p85 subunit of PI 3-kinase during cell adhesion and observed direct phosphorylation of p85 by FAK in vitro. Together, these results suggest that PI 3-kinase may be a FAK substrate in vivo and serve as an effector of FAK.     

c-Met represents a potential therapeutic target for personalized treatment in hepatocellular carcinoma

c-Met, a high-affinity receptor for hepatocyte growth factor (HGF), plays a critical role in cancer growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with an active HGF/c-Met signaling pathway have a significantly worse prognosis. Although targeting the HGF/c-Met pathway has been proposed for the treatment of multiple cancers, the effect of c-Met inhibition in HCC remains unclear. The human HCC cell lines Huh7, Hep3B, MHCC97-L, and MHCC97-H were used in this study to investigate the effect of c-Met inhibition using the small molecule selective c-Met tyrosine kinase inhibitor PHA665752. MHCC97-L and MHCC97-H cells demonstrate a mesenchymal phenotype with decreased expression of E-cadherin and increased expression of c-Met, fibronectin, and Zeb2 compared with Huh7 and Hep3B cells, which have an epithelial phenotype. PHA665752 treatment blocked phosphorylation of c-Met and downstream phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase/Erk pathways, inhibited cell proliferation, and induced apoptosis in c-Met-positive MHCC97-L and MHCC97-H cells. In xenograft models, administration of PHA665752 significantly inhibited c-Met-positive MHCC97-L and MHCC97-H tumor growth, and PHA665752-treated tumors demonstrated marked reduction of both c-Met phosphorylation and cell proliferation. c-Met-negative Huh7 and Hep3B cells were not affected by c-Met inhibitor treatment in vitro or in vivo. In addition, c-Met-positive MHCC97-L and MHCC97-H cells demonstrated cancer stem cell-like characteristics, such as resistance to chemotherapy, tumor sphere formation, and increased expression of CD44 and ABCG2, and PHA665752 treatment suppressed tumor sphere formation and inhibited CD44 expression. Conclusion: c-Met represents a potential target of personalized treatment for HCC with an active HGF/c-Met pathway.     

Mediation of basic fibroblast growth factor-induced lactotropic cell proliferation by Src-Ras-mitogen-activated protein kinase p44/42 signaling

Basic fibroblast growth factor (bFGF), which is secreted from folliculostellate cells in the anterior pituitary, is known to be involved in the communication between folliculostellate cells and lactotropes during estradiol-induced lactotropic cell proliferation. We studied the role of MAPK p44/42 in bFGF-regulated cell proliferation using enriched lactotropes and the lactotrope-derived PR1 cell line. In cell cultures, bFGF increased cell proliferation of PR1 cells and enriched lactotropes. In both of these cell populations, bFGF also increased phosphorylation of MAPK p44/42. U0126, an inhibitor of MAPK p44/42, blocked the bFGF-induced activation of MAPK p44/42 as well as the bFGF-induced cell proliferation of enriched lactotropes and PR1 cells. Treatment of PR1 cells with bFGF increased the activity of Ras p21, whereas overexpression of a dominant negative mutant of Ras p21 abrogated the bFGF-induced activation of MAPK p44/42 in these cells. Furthermore, the Src kinase inhibitor PP1 suppressed bFGF-induced activation of MAPK p44/42 in both enriched lactotropes and PR1 cells. The Src kinase inhibitor PP1 also reduced bFGF activation of Ras p21 and cell proliferation in PR1 cells. On the other hand, the bFGF-induced activation of MAPK p44/42 in enriched lactotropes and PR1 cells was not affected by protein kinase C inhibitors. These data suggest that bFGF induction of lactotropic cell proliferation is possibly mediated by activation of Src kinase, Ras p21, and MAPK p44/42.     

Cell surface proteoglycan syndecan-1 mediates hepatocyte growth factor binding and promotes Met signaling in multiple myeloma

Heparan sulfate proteoglycans (HSPGs) play a crucial role in growth regulation by assembling signaling complexes and presenting growth factors to their cognate receptors. Within the immune system, expression of the HSPG syndecan-1 (CD138) is characteristic of terminally differentiated B cells, ie, plasma cells, and their malignant counterpart, multiple myeloma (MM). This study explored the hypothesis that syndecan-1 might promote growth factor signaling and tumor growth in MM. For this purpose, the interaction was studied between syndecan-1 and hepatocyte growth factor (HGF), a putative paracrine and autocrine regulator of MM growth. The study demonstrates that syndecan-1 is capable of binding HGF and that this growth factor is indeed a potent stimulator of MM survival and proliferation. Importantly, the interaction of HGF with heparan sulfate moieties on syndecan-1 strongly promotes HGF-mediated signaling, resulting in enhanced activation of Met, the receptor tyrosine kinase for HGF. Moreover, HGF binding to syndecan-1 promotes activation of the phosphatidylinositol 3-kinase/protein kinase B and RAS/mitogen-activated protein kinase pathways, signaling routes that have been implicated in the regulation of cell survival and proliferation, respectively. These results identify syndecan-1 as a functional coreceptor for HGF that promotes HGF/Met signaling in MM cells, thus suggesting a novel function for syndecan-1 in MM tumorigenesis.