c-Src Signaling Pathways Research Articles

Cryptocaryone induces apoptosis in human hepatocellular carcinoma cells by inhibiting aerobic glycolysis through Akt and c-Src signaling pathways.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, with the second highest mortality rate in all cancer. Energy reprogramming is one of the hallmarks of cancer, and emerging evidence showed that targeting glycolysis is a promising strategy for HCC treatment. Cryptocaryone has been shown to display promising anti-cancer activity against numerous types of cancer. Previous study also indicated that cryptocaryone induces cytotoxicity by inhibiting glucose transport in cancer cells, but the detailed mechanism still needs to be elucidated. Therefore, this study aimed to investigate the relationship between the anti-cancer effect and glycolytic metabolism of cryptocaryone in human HCC cells. In this study, we found that cryptocaryone potently induced growth inhibition by apoptotic cell death in HCC cells. Cryptocaryone also suppressed the ATP synthesis, lactate production and glycolytic capacity of HCC cells. Mechanistic investigations showed that phosphorylation of Akt and c-Src, as well as the expression of HK1 were impeded by cryptocaryone. Moreover, cryptocaryone markedly increased the expression level of transcription factor FoxO1. Importantly, clinical database analysis confirmed the negative correlation between HK1 and FoxO1. High expression levels of HK-1 were positively correlated with poorer survival in patients with HCCs. These results suggest that cryptocaryone may promote cell apoptosis by inhibiting FoxO1-mediated aerobic glycolysis through Akt and c-Src signaling cascades in human HCC cells. This is the first study to indicate that cryptocaryone exerts anti-cancer property against human HCC cells. Cryptocaryone is a potential natural product worthy of further development into a promising candidate for HCC treatment.

Active Gαi/o Mutants Accelerate Breast Tumor Metastasis via the c-Src Pathway.

Constitutively active mutations in the Gαi2 and GαoA subunits of heterotrimeric G proteins have been found in various human cancers, including breast cancer, but their precise roles in tumor formation, progression, and metastasis remain poorly understood. This study focused on GαoAR243H and Gαi2R179C mutants in breast cancer. These mutants alone were insufficient to initiate mammary tumor formation in mice. However, when introduced into transgenic mouse models of breast cancer induced by Neu expression or PTEN loss, the Gαi2R179C mutant notably enhanced spontaneous lung metastasis, without affecting primary tumor initiation and growth. Ectopic expression of the GαoAR243H and Gαi2R179C mutants in tumor cells promoted cell migration invitro and dissemination into multiple organs invivo by activating the c-Src signaling pathway. These mutants activate c-Src through direct interaction, involving specific residues in the switch domains II of Gαi subunits, which only partially overlap with those involved in inhibiting adenylyl cyclases. This study uncovers a critical role of Gαi/o signaling in accelerating breast cancer metastasis through the c-Src pathway. These findings hold clinical significance as they may pave the way for personalized therapies targeting c-Src to inhibit breast cancer metastasis in patients with active Gαi/o mutations or elevated Gαi/o signaling.

FGF2 drives osteosarcoma metastasis through activating FGFR1-4 receptor pathway-mediated ICAM-1 expression

Osteosarcoma is a malignant tumor with high metastatic potential, such that the overall 5-year survival rate of patients with metastatic osteosarcoma is only 20%. Therefore, it is necessary to unravel the mechanisms of osteosarcoma metastasis to identify predictors of metastasis by which to develop new therapies. Fibroblast growth factor 2 (FGF2) is a growth factor involved in embryonic development, cell migration, and proliferation. The overexpression of FGF2 and FGF receptors (FGFRs) has been shown to enhance cancer cell proliferation in lung, breast, gastric, and prostate cancers as well as melanoma. Nonetheless, the roles of FGF2 and FGFRs in human osteosarcoma cells remain unknown. In the present study, we found that FGF2 was overexpressed in human osteosarcoma sections and correlated with lung metastasis. Treatment of FGF2 induced migration activity, invasion activity, and intercellular adhesion molecule (ICAM)-1 expression in osteosarcoma cells. In particular, the downregulation or antagonism of FGFR1-4 suppressed FGF2-induced ICAM-1 expression and cancer cell migration. Furthermore, FGFR1, FGFR2, FGFR3, and FGFR4 were involved in FGF2-induced the phospholipase Cβ/protein kinase Cα/proto-oncogene c-Src signaling pathway and triggered c-Jun nuclear translocation. Subsequent c-Jun upregulation of activator protein-1 transcription activity on the ICAM-1 promoter led to an increased migration of osteosarcoma cells. Moreover, the knockdown of endogenous FGF2 suppressed ICAM-1 expression and migration of osteosarcoma cells. These findings suggest that FGF2/FGFR1-4 signaling promotes metastasis via its direct downstream target gene ICAM-1, revealing a novel potential therapeutic target for osteosarcoma.

CXCL14 promotes metastasis of non-small cell lung cancer through ACKR2-depended signaling pathway.

Background: Lung cancer is a malignant tumor with metastatic potential. Chemokine ligand 14 (CXCL14) has been reported to be associated with different cancer cell migration and invasion. However, few studies have explored the function of CXCL14 and its specific receptor in lung cancer metastasis. This study aims to determine the mechanism of CXCL14-promoted cancer metastasis. Methods: The expression of CXCL14, atypical chemokine receptor 2 (ACKR2), and epithelial mesenchymal transition (EMT) markers was evaluated by the public database of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), Western blot, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry (IHC), and immunofluorescence (IF). Migration and wound healing assays were used to observe the motility of cancer cells. A luciferase reporter assay was performed to analyze transcription factor activity. The metastasis of lung cancer cells was evaluated in an orthotopic model. Results: We have presented that overexpression of CXCL14 and ACKR2 was observed in lung cancer datasets, human lung tumor sections, and lung cancer cells. Furthermore, the migration of CXCL14-promoted lung cancer cells was determined in vitro and in vivo. In particular, ACKR2 knockdown abolished CXCL14-induced cancer cell motility. Additionally, ACKR2 was involved in CXCL14-triggered phospholipase Cβ3 (PLCβ3), protein kinase Cα (PKCα), and proto-oncogene c-Src signaling pathway and subsequently upregulated nuclear factor κB (NF-κB) transcription activity leading to EMT and migration of lung cancer cells. These results indicated that the CXCL14/ACKR2 axis played an important role in lung cancer metastasis. Conclusion: This study is the first to reveal the function of CXCL14 in promoting EMT and metastasis in lung cancer. As a specific receptor for CXCL14 in lung cancer, ACKR2 mediates CXCL14-induced signaling that leads to cell motility. Our findings can be used as a prognostic biomarker of lung cancer metastasis.

Interplay between c-Src and the APC/C co-activator Cdh1 regulates mammary tumorigenesis

The Anaphase Promoting Complex (APC) coactivator Cdh1 drives proper cell cycle progression and is implicated in the suppression of tumorigenesis. However, it remains elusive how Cdh1 restrains cancer progression and how tumor cells escape the inhibition of Cdh1. Here we report that Cdh1 suppresses the kinase activity of c-Src in an APC-independent manner. Depleting Cdh1 accelerates breast cancer cell proliferation and cooperates with PTEN loss to promote breast tumor progression in mice. Hyperactive c-Src, on the other hand, reciprocally inhibits the ubiquitin E3 ligase activity of APCCdh1 through direct phosphorylation of Cdh1 at its N-terminus, which disrupts the interaction between Cdh1 and the APC core complex. Furthermore, pharmacological inhibition of c-Src restores APCCdh1 tumor suppressor function to repress a panel of APCCdh1 oncogenic substrates. Our findings reveal a reciprocal feedback circuit of Cdh1 and c-Src in the crosstalk between the cell cycle machinery and the c-Src signaling pathway.

RANKL-induced c-Src activation contributes to conventional anti-cancer drug resistance and dasatinib overcomes this resistance in RANK-expressing multiple myeloma cells.

The survival and growth of multiple myeloma (MM) cells are facilitated by cell-cell interactions with bone marrow stromal cells and the bone marrow microenvironment. These interactions induce de novo drug resistance known as cell adhesion-mediated drug resistance. Our previous results recently revealed that the receptor activator of NF-κB (RANK) ligand (RANKL), which is expressed by bone marrow stromal cells, contributes to anti-cancer drug resistance through the activation of various signaling molecules and suppression of Bim expression in RANK-expressing MM cells. However, the detailed mechanisms underlying RANKL-induced drug resistance remain uncharacterized. In the present study, we investigated the mechanism of RANKL-induced drug resistance in RANK-expressing MM cell lines. We found treatment of MM cells with RANKL-induced c-Src phosphorylation and activation of the downstream signaling molecules Akt, mTOR, STAT3, JNK, and NF-κB. In addition, treatment with dasatinib, a c-Src inhibitor, overcame RANKL- and bone marrow stromal cell-induced drug resistance to adriamycin, vincristine, dexamethasone, and melphalan by suppressing c-Src, Akt, mTOR, STAT3, JNK, and NF-κB activation and enhancing expression of Bim. Overall, RANKL- and bone marrow stromal cell-induced drug resistance correlated with the activation of c-Src signaling pathways, which caused a decrease in Bim expression. Dasatinib treatment of RANK-expressing MM cells re-sensitized them to anti-cancer drugs. Therefore, inhibition of c-Src may be a new therapeutic approach for overcoming RANKL-induced drug resistance in patients with MM.

Resistin facilitates VEGF-C-associated lymphangiogenesis by inhibiting miR-186 in human chondrosarcoma cells

Chondrosarcoma is a common primary malignant tumor of the bone that can metastasize through the vascular system to other organs. A key step in the metastatic process, lymphangiogenesis, involves vascular endothelial growth factor-C (VEGF-C). However, the effects of lymphangiogenesis in chondrosarcoma metastasis remain to be clarified. Accumulating evidence shows that resistin, a cytokine secreted from adipocytes and monocytes, also promotes tumor pathogenesis. Notably, chondrosarcoma can easily metastasize. In this study, we demonstrate that resistin enhances VEGF-C expression and lymphatic endothelial cells (LECs)-associated lymphangiogenesis in human chondrosarcoma cells. We also show that resistin triggers VEGF-C-dependent lymphangiogenesis via the c-Src signaling pathway and down-regulating micro RNA (miR)-186. Overexpression of resistin in chondrosarcoma cells significantly enhanced VEGF-C production and LECs-associated lymphangiogenesis in vitro and tumor-related lymphangiogenesis in vivo. Resistin levels were positively correlated with VEGF-C-dependent lymphangiogenesis via the down-regulation of miR-186 expression in clinical samples from chondrosarcoma tissue. This study is the first to evaluate the mechanism underlying resistin-induced promotion of LECs-associated lymphangiogenesis via the upregulation of VEGF-C expression in human chondrosarcomas. We suggest that resistin may represent a molecular target in VEGF-C-associated tumor lymphangiogenesis in chondrosarcoma metastasis.

Increased PTPRA expression leads to poor prognosis through c-Src activation and G1 phase progression in squamous cell lung cancer.

PTPRA is reported to be involved in cancer development and progression through activating the Src family kinase (SFK) signaling pathways, however, the roles of PTPRA in the squamous cell lung cancer (SCC) development are unclear. The purpose of this study was to clarify the clinical relevance and biological roles of PTPRA in SCC. We found that PTPRA was upregulated in squamous cell lung cancer compared to matched normal tissues at the mRNA (N=20, P=0.004) and protein expression levels (N=75, P<0.001). Notably, high mRNA level of PTPRA was significantly correlated with poorer prognosis in 675 SCC patients from the Kaplan-Meier plotter database. With 75 cases, we found that PTPRA protein expression was significantly correlated with tumor size (P=0.002), lymph node metastasis (P=0.008), depth of tumor invasion (P<0.001) and clinical stage (P<0.001). The Kaplan-Meier plot suggested that high expression of PTPRA had poorer overall survival in SCC patients (P=0.009). Multivariate Cox regression analysis suggested that PTPRA expression was an independent prognostic factor in SCC patients. In the cellular models, PTPRA promotes SCC cell proliferation through modulating Src activation as well as cell cycle progression. In conclusion, higher PTPRA level was associated with worse prognosis of SCC patients and PTPRA could promote the cell cycle progression through stimulating the c-Src signaling pathways.

Basic fibroblast growth factor promotes VEGF-C-dependent lymphangiogenesis via inhibition of miR-381 in human chondrosarcoma cells.

A chondrosarcoma is a common, primary malignant bone tumor that can grow to destroy the bone, produce fractures and develop soft tissue masses. Left untreated, chondrosarcomas metastasize through the vascular system to the lungs and ultimately lead to large metastatic deposits of the malignant cartilage taking over lung volume and function. Vascular endothelial growth factor (VEGF)-C has been implicated in tumor-induced lymphangiogenesis and elevated expression of VEGF-C has been found to correlate with cancer metastasis. bFGF (basic fibroblast growth factor), a secreted cytokine, regulates biological activity, including angiogenesis and metastasis. We have previously reported on the important role of bFGF in angiogenesis in chondrosarcomas. However, the effect of bFGF in VEGF-C regulation and lymphangiogenesis in chondrosarcomas is poorly understood. In this investigation, we demonstrate a correlation exists between bFGF and VEGF-C in tissue specimens from patients with chondrosarcomas. To examine the lymphangiogenic effect of bFGF, we used human lymphatic endothelial cells (LECs) to mimic lymphatic vessel formation. We found that bFGF-treated chondrosarcomas promoted LEC tube formation and cell migration. In addition, bFGF knockdown inhibited lymphangiogenesis in vitro and in vivo. We also found that bFGF-induced VEGF-C is mediated by the platelet-derived growth factor receptor (PDGFR) and c-Src signaling pathway. Furthermore, bFGF inhibited microRNA-381 expression via the PDGFR and c-Src cascade. Our study is the first to describe the mechanism of bFGF-promoted lymphangiogenesis by upregulating VEGF-C expression in chondrosarcomas. Thus, bFGF could serve as a therapeutic target in chondrosarcoma metastasis and lymphangiogenesis.

Abstract P1-07-06: Activation of oncogenic pathways by mitochondrial reprogramming in triple negative breast cancer

Abstract Triple negative breast cancer (TN BCa) Driver pathway is still poorly understood. Thus, it is important to identify the underlying mechanisms of triple negative breast cancer progression. Mitochondria, a semiautonomous organelle in cells, play an important role in cellular energy metabolism, free radical generation, and apoptosis. Mitochondria-nuclear crosstalk is a bidirectional pathway of communication between mitochondria and nucleus that influences many cellular and organismal activities. This crosstalk can regulate several oncogenic pathways involved in tumorigenesis. Using transmitochondrial cybrid (cybrid) technology, we generated different cybrid models under common nuclear background of TN BCa. Mitochondria from cells of different cancer potential including benign breast epithelium, moderately and highly metastatic breast cancer cell lines were used to understand cancer mitochondria regulated tumor pathways. Tumor and gene expression analysis suggested that among different cancer pathways, c-Src signaling pathway is one of the most consistently activated pathways in cybrids with TN BCa cancer mitochondria. Further analysis in parental cells and other tumor models suggested that autophosphorylation of c-Src is regulated by mitochondrial tumor characteristics. Our preliminary analysis also suggest that mitochondria targeted drugs are promising combination therapy for the management of Src-driven TN BCa. This finding is particularly important while considering the poor response rate observed after single drug therapy with Src family tyrosine kinase inhibitor Dasatinib in unselected TN BCa patients. Citation Format: Jun Hyoung Park, Santhosh Kumar, Sajna Vithayathil, Kavisha Arora, Nagireddy Putluri, Efrosini Tsouko, Taraka R Donti, Daniel E Frigo, Chad J Creighton, Michael T Lewis, Arun Sreekumar, Lee-Jun Wong, Benny Abraham Kaipparettu. Activation of oncogenic pathways by mitochondrial reprogramming in triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-06.

Paeonol suppresses chondrosarcoma metastasis through up-regulation of miR-141 by modulating PKCδ and c-Src signaling pathway.

Chondrosarcoma, a primary malignant bone cancer, has potential for local invasion and distant metastasis, especially to the lungs. Patients diagnosed with it show poor prognosis. Paeonol (2'-hydroxy-4'-methoxyacetophenone), the main active compound of traditional Chinese remedy Paeonia lactiflora Pallas, exhibits anti-inflammatory and anti-tumor activity; whether paeonol regulates metastatic chondrosarcoma is largely unknown. Here, we find paeonol do not increase apoptosis. By contrast, at non-cytotoxic concentrations, paeonol suppresses migration and invasion of chondrosarcoma cells. We also demonstrate paeonol enhancing miR-141 expression and miR-141 inhibitor reversing paeonol-inhibited cell motility; paeonol also reduces protein kinase C (PKC)δ and c-Src kinase activity. Since paeonol inhibits migration and invasion of human chondrosarcoma via up-regulation of miR-141 via PKCδ and c-Src pathways, it thus might be a novel anti-metastasis agent for treatment of metastatic chondrosarcoma.

ER-α36-mediated gastric cancer cell proliferation via the c-Src pathway

Previously, a novel variant of estrogen receptor (ER)-α, ER-α36, was identified and cloned and reported to mainly mediate non-genomic estrogen signaling. More recently, we identified that ER-α36 is important for the invasion and lymph node metastasis of human gastric cancer. In the present study, the c-Src signaling pathway was demonstrated to be involved in the non-genomic estrogen signaling mediated by ER-α36 in SGC7901 gastric cancer cells. SGC7901 cells were subjected to the siRNA-mediated knockdown of ER-α36 (PLKO.1-PURO-SP6-ER-α36-L) or transfected with an ER-α36 upregulated expression plasmid (PLJM1-ER-α36-H) and treated with 17β-estradiol (E2β) and PP2, a c-Src protein inhibitor. The expression of ER-α36 and c-src/p-c-Src and cyclin D1 was examined by western blot analysis, and tumor cell growth was analyzed by cell proliferation and nude mouse xenograft assays. The ER variant, ER-α36, was shown to enhance gastric cancer cell proliferation through activation of the membrane-initiated c-Src signaling pathways, indicating that ER-α36 is important for the regulation of proliferation in gastric cancer. In addition, ER-α36 was shown to directly interact with c-Src by immunoprecipitation. The results of the present study indicate that the use of ER-α36 may be a targeted therapeutic approach in gastric cancer.

Targeted disruption of leucine-rich repeat kinase 1 but not leucine-rich repeat kinase 2 in mice causes severe osteopetrosis

To assess the roles of Lrrk1 and Lrrk2, we examined skeletal phenotypes in Lrrk1 and Lrrk2 knockout (KO) mice. Lrrk1 KO mice exhibit severe osteopetrosis caused by dysfunction of multinucleated osteoclasts, reduced bone resorption in endocortical and trabecular regions, and increased bone mineralization. Lrrk1 KO mice have lifelong accumulation of bone and respond normally to the anabolic actions of teriparatide treatment, but are resistant to ovariectomy-induced bone boss. Precursors derived from Lrrk1 KO mice differentiate into multinucleated cells in response to macrophage colony-stimulating factor (M-CSF)/receptor activator of NF-κB ligand (RANKL) treatment, but these cells fail to form peripheral sealing zones and ruffled borders, and fail to resorb bone. The phosphorylation of cellular Rous sarcoma oncogene (c-Src) at Tyr-527 is significantly elevated whereas at Tyr-416 is decreased in Lrrk1-deficient osteoclasts. The defective osteoclast function is partially rescued by overexpression of the constitutively active form of Y527F c-Src. Immunoprecipitation assays in osteoclasts detected a physical interaction of Lrrk1 with C-terminal Src kinase (Csk). Lrrk2 KO mice do not show obvious bone phenotypes. Precursors derived from Lrrk2 KO mice differentiate into functional multinucleated osteoclasts. Our finding of osteopetrosis in Lrrk1 KO mice provides convincing evidence that Lrrk1 plays a critical role in negative regulation of bone mass in part through modulating the c-Src signaling pathway in mice.

Regulation of Caveolin-1 Expression and Phosphorylation by VEGF in Ovine Amnion Cells

Vascular endothelial growth factor (VEGF) has been implicated in the regulation of vesicular transport of amniotic fluid via caveolae across the amnion. This study tested the hypothesis that VEGF regulates caveolar function by stimulating caveolin-1 expression and phosphorylation in ovine amniotic epithelial cells (oAECs). Using primary cultures of oAECs, caveolin-1 was identified by immunofluorescent staining. Caveolin-1 messenger RNA (mRNA) abundance was determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and protein by Western blotting. The effects of VEGF( 165) on caveolin-1 expression and phosphorylation were determined. Caveolin-1 immunoreactivity was detected in oAECs. In response to 10 ng/mL VEGF( 165), caveolin-1 mRNA levels increased whereas the protein levels were unaffected. Furthermore, VEGF stimulated caveolin-1 phosphorylation, an effect abrogated by the inhibition of c-Src protein kinase. These data suggest that VEGF upregulates caveolin-1 activity through c-Src signaling pathways. Our observations support the hypothesis that VEGF regulates amniotic fluid transport across the amnion by stimulating caveolin-1 activity to mediate caveolar function in amnion cells.

E-Cadherin Adhesion Activates c-Src Signaling at Cell–Cell Contacts

Cadherin-based cell-cell contacts are prominent sites for phosphotyrosine signaling, being enriched in tyrosine-phosphorylated proteins and tyrosine kinases and phosphatases. The functional interplay between cadherin adhesion and tyrosine kinase signaling, however, is complex and incompletely understood. In this report we tested the hypothesis that cadherin adhesion activates c-Src signaling and sought to assess its impact on cadherin function. We identified c-Src as part of a cadherin-activated cell signaling pathway that is stimulated by ligation of the adhesion receptor. However, c-Src has a biphasic impact on cadherin function, exerting a positive supportive role at lower signal strengths, but inhibiting function at high signal strengths. Inhibiting c-Src under circumstances when it is activated by cadherin adhesion decreased several measures of cadherin function. This suggests that the cadherin-activated c-Src signaling pathway serves positively to support cadherin function. Finally, our data implicate PI3-kinase signaling as a target for cadherin-activated c-Src signaling that contributes to its positive impact on cadherin function. We conclude that E-cadherin signaling is an important activator of c-Src at cell-cell contacts, providing a key input into a signaling pathway where quantitative changes in signal strength may result in qualitative differences in functional outcome.

MUC1 cytoplasmic tail: a potential therapeutic target for ovarian carcinoma

Ovarian cancer is often a lethal disease, since the occult progression of the tumor within the peritoneal cavity results in late diagnosis and treatment failure. The identification of molecular events specific to metastasis is critical for the development of effective therapies. MUC1 is aberrantly overexpressed by most ovarian cancer and regarded as a molecular target for ovarian cancer. This review focuses on the latest advances regarding a signaling region in the MUC1 C-terminal subunit-mediated c-Src signaling pathways in malignant transformation, invasion and metastasis. Disruption of MUC1-C-terminal subunit-associated c-Src signaling by targeting the specific sites might represent a novel immunotherapeutic approach for the treatment of ovarian cancer.

Cryptosporidium parvum invasion of biliary epithelia requires host cell tyrosine phosphorylation of cortactin via c-Src

Background & aims: Cryptosporidium parvum invasion of epithelia requires polymerization of host cell actin at the attachment site. We analyzed the role of host cell c-Src, a cytoskeleton-associated protein tyrosine kinase, in C. parvum invasion of biliary epithelia. Methods: In vitro models of biliary cryptosporidiosis using a human biliary epithelial cell line were used to assay the role of c-Src signaling pathway in C. parvum invasion. Results: c-Src and cortactin, an actin-binding protein and a substrate for c-Src, were recruited to the parasite-host cell interface during C. parvum invasion. Tyrosine phosphorylation of cortactin in infected cells was also detected. Inhibition of host cell c-Src significantly blocked C. parvum-induced accumulation and tyrosine phosphorylation of cortactin and actin polymerization at the attachment sites, thereby inhibiting C. parvum invasion of biliary epithelial cells. A triple mutation of tyrosine of cortactin in the epithelia also diminished C. parvum invasion. In addition, proteins originating from the parasite were detected within infected cells at the parasite-host cell interface. Antiserum against C. parvum membrane proteins blocked accumulation of c-Src and cortactin and significantly decreased C. parvum invasion. No accumulation of the endocytosis-related proteins, dynamin 2 and clathrin, was found at the parasite-host cell interface; also, inhibition of dynamin 2 did not block C. parvum invasion. Conclusions: C. parvum invasion of biliary epithelial cells requires host cell tyrosine phosphorylation of cortactin by a c-Src-mediated signaling pathway to induce actin polymerization at the attachment site, a process associated with microbial secretion but independent of host cell endocytosis.

The Differentiation of Skeletal Muscle Cells Involves a Protein-tyrosine Phosphatase-α-mediated C-Src Signaling Pathway

Protein-tyrosine phosphatase-alpha (PTPalpha) plays an important role in various cellular signaling events, including proliferation and differentiation. In this study, we established L6 cell lines either underexpressing or overexpressing PTPalpha by stable transfection of cells with antisense PTPalpha or with full-length wild-type human or mouse or double catalytic site Cys --> Ala mutant (DM8) PTPalpha cDNA. Expression of PTPalpha in these cell lines was determined by immunoblotting and immunofluorescence. Cells harboring antisense PTPalpha exhibited a significantly reduced growth rate and thymidine incorporation when compared with the wild-type L6 cells. In contrast, cells overexpressing PTPalpha showed more rapid (2-fold) proliferation. Myoblasts with diminished PTPalpha failed to undergo fusion and did not form myotubes in reduced serum whereas overexpression of PTPalpha promoted myogenesis 2 days earlier than wild-type L6 cells. Overexpression of phosphatase-inactive mutant PTPalpha recapitulated the phenotype of the antisense cells. The different myogenic activities of these cell lines were correlated with the expression of myogenin and creatine kinase activity. Consistent with previous reports, PTPalpha positively regulated the activity of the protein-tyrosine kinase Src. Treatment of L6 cells with PP2 or SU6656, specific inhibitors of Src family kinases, and transient transfection of dominant-inhibitory Src inhibited the formation of myotubes and expression of myogenin. Moreover, enhanced expression of PTPalpha and activation of Src was detected during myogenesis. Together, these data indicate that PTPalpha is involved in the regulation of L6 myoblast growth and skeletal muscle cell differentiation via an Src-mediated signaling pathway.