Fibroblast Growth Requires CT10 Regulator of Kinase (Crk) and Crk-like (CrkL)
2016; Elsevier BV; Volume: 291; Issue: 51 Linguagem: Inglês
10.1074/jbc.m116.764613
ISSN1083-351X
AutoresTae‐Ju Park, Mateusz Koptyra, Tom Curran,
Tópico(s)RNA Interference and Gene Delivery
ResumoCT10 regulator of kinase (Crk) and Crk-like (CrkL) are the cellular counterparts of the viral oncogene v-Crk. Elevated levels of Crk and CrkL have been observed in many human cancers; inhibition of Crk and CrkL expression reduced the tumor-forming potential of cancer cell lines. Despite a close relationship between the Crk family proteins and tumorigenesis, how Crk and CrkL contribute to cell growth is unclear. We ablated endogenous Crk and CrkL from cultured fibroblasts carrying floxed alleles of Crk and CrkL by transfection with synthetic Cre mRNA (synCre). Loss of Crk and CrkL induced by synCre transfection blocked cell proliferation and caused shrinkage of the cytoplasm and the nucleus, formation of adherens junctions, and reduced cell motility. Ablation of Crk or CrkL alone conferred a much more modest reduction in cell proliferation. Reintroduction of CrkI, CrkII, or CrkL individually rescued cell proliferation in the absence of the endogenous Crk and CrkL, suggesting that Crk and CrkL play overlapping functions in regulating fibroblast growth. Serum and basic FGF induced phosphorylation of Akt, MAP kinases, and S6 kinase and Fos expression in the absence of Crk and CrkL, suggesting that cells lacking Crk and CrkL are capable of initiating major signal transduction pathways in response to extracellular stimuli. Furthermore, cell cycle and cell death analyses demonstrated that fibroblasts lacking Crk and CrkL become arrested at the G1-S transition and undergo a modest apoptosis. Taken together, our results suggest that Crk and CrkL play essential overlapping roles in fibroblast growth. CT10 regulator of kinase (Crk) and Crk-like (CrkL) are the cellular counterparts of the viral oncogene v-Crk. Elevated levels of Crk and CrkL have been observed in many human cancers; inhibition of Crk and CrkL expression reduced the tumor-forming potential of cancer cell lines. Despite a close relationship between the Crk family proteins and tumorigenesis, how Crk and CrkL contribute to cell growth is unclear. We ablated endogenous Crk and CrkL from cultured fibroblasts carrying floxed alleles of Crk and CrkL by transfection with synthetic Cre mRNA (synCre). Loss of Crk and CrkL induced by synCre transfection blocked cell proliferation and caused shrinkage of the cytoplasm and the nucleus, formation of adherens junctions, and reduced cell motility. Ablation of Crk or CrkL alone conferred a much more modest reduction in cell proliferation. Reintroduction of CrkI, CrkII, or CrkL individually rescued cell proliferation in the absence of the endogenous Crk and CrkL, suggesting that Crk and CrkL play overlapping functions in regulating fibroblast growth. Serum and basic FGF induced phosphorylation of Akt, MAP kinases, and S6 kinase and Fos expression in the absence of Crk and CrkL, suggesting that cells lacking Crk and CrkL are capable of initiating major signal transduction pathways in response to extracellular stimuli. Furthermore, cell cycle and cell death analyses demonstrated that fibroblasts lacking Crk and CrkL become arrested at the G1-S transition and undergo a modest apoptosis. Taken together, our results suggest that Crk and CrkL play essential overlapping roles in fibroblast growth. The chicken tumor virus oncoprotein, v-Crk, induces a substantial increase in protein tyrosine phosphorylation through protein-protein interactions mediated by its SH2 2The abbreviations used are: SH2 and SH3, Src homology domains 2 and 3; CrkL, Crk (CT10 regulator of kinase)-like; synCre, synthetic Cre mRNA; synRNA, synthetic mRNA; DPT, day(s) post transduction; synGFP, synthetic green fluorescent protein mRNA; HSP90, heat shock protein 90; ROI, region of interest. and SH3 domains, leading to cell transformation (1.Mayer B.J. Hamaguchi M. Hanafusa H. Characterization of p47gag-crk, a novel oncogene product with sequence similarity to a putative modulatory domain of protein-tyrosine kinases and phospholipase C.Cold Spring Harb. Symp. Quant. Biol. 1988; 53: 907-914Crossref PubMed Google Scholar). CrkII and Crk-like (CrkL) represent closely related cellular homologues of v-Crk and have similar structures and functions (2.Kumar S. Fajardo J.E. Birge R.B. Sriram G. Crk at the quarter century mark: perspectives in signaling and cancer.J. Cell. Biochem. 2014; 115: 819-825Crossref PubMed Scopus (26) Google Scholar). CrkI is a splice variant of CrkII that lacks the C-terminal SH3 domain. Crk family proteins (CrkI, CrkII, and CrkL) interact with many proteins through their SH2 and SH3 domains. Overexpression of Crk family proteins induces transformation of cultured cells (3.Matsuda M. Tanaka S. Nagata S. Kojima A. Kurata T. Shibuya M. Two species of human CRK cDNA encode proteins with distinct biological activities.Mol. Cell. Biol. 1992; 12: 3482-3489Crossref PubMed Scopus (250) Google Scholar, 4.Senechal K. Halpern J. Sawyers C.L. The CRKL adaptor protein transforms fibroblasts and functions in transformation by the BCR-ABL oncogene.J. Biol. Chem. 1996; 271: 23255-23261Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). 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Adaptor molecule Crk is required for sustained phosphorylation of Grb2-associated binder 1 and hepatocyte growth factor-induced cell motility of human synovial sarcoma cell lines.Mol Cancer Res. 2006; 4: 499-510Crossref PubMed Scopus (56) Google Scholar), glioblastoma (14.Wang L. Tabu K. Kimura T. Tsuda M. Linghu H. Tanino M. Kaneko S. Nishihara H. Tanaka S. Signaling adaptor protein Crk is indispensable for malignant feature of glioblastoma cell line KMG4.Biochem. Biophys. Res. Commun. 2007; 362: 976-981Crossref PubMed Scopus (36) Google Scholar), breast cancer (10.Fathers K.E. Bell E.S. Rajadurai C.V. Cory S. Zhao H. Mourskaia A. Zuo D. Madore J. Monast A. Mes-Masson A.M. Grosset A.A. Gaboury L. Hallet M. Siegel P. Park M. Crk adaptor proteins act as key signaling integrators for breast tumorigenesis.Breast Cancer Res. 2012; 14: R74Crossref PubMed Scopus (51) Google Scholar), head and neck squamous cell carcinoma (17.Yanagi H. Wang L. Nishihara H. Kimura T. Tanino M. Yanagi T. Fukuda S. Tanaka S. CRKL plays a pivotal role in tumorigenesis of head and neck squamous cell carcinoma through the regulation of cell adhesion.Biochem. Biophys. Res. Commun. 2012; 418: 104-109Crossref PubMed Scopus (22) Google Scholar), and rhabdomyosarcoma (18.Yeung C.L. Ngo V.N. Grohar P.J. Arnaldez F.I. Asante A. Wan X. Khan J. Hewitt S.M. Khanna C. Staudt L.M. Helman L.J. Loss-of-function screen in rhabdomyosarcoma identifies CRKL-YES as a critical signal for tumor growth.Oncogene. 2013; 32: 5429-5438Crossref PubMed Scopus (36) Google Scholar) cell lines. Taken together, these reports imply that elevated levels of Crk family proteins promote cell transformation and enhance tumor cell growth (for review see Refs. 19.Sriram G. Birge R.B. Emerging roles for crk in human cancer.Genes Cancer. 2010; 1: 1132-1139Crossref PubMed Scopus (52) Google Scholar and 20.Tsuda M. Tanaka S. Roles for crk in cancer metastasis and invasion.Genes Cancer. 2012; 3: 334-340Crossref PubMed Scopus (38) Google Scholar). To investigate functions of endogenous Crk and CrkL in biological processes at the cellular level, we developed mouse strains and cell lines harboring individual and combined floxed alleles of Crk and CrkL. Application of the Cre-loxP recombination-based conditional knock-out approach to cultured fibroblasts enabled us to demonstrate that endogenous Crk and CrkL are important for maintaining cell structural integrity and proper cell motility (21.Park T.J. Curran T. Essential roles of Crk and CrkL in fibroblast structure and motility.Oncogene. 2014; 33: 5121-5132Crossref PubMed Scopus (29) Google Scholar). We also discovered that Crk and CrkL are required for cell transformation induced by viral oncogenes (22.Koptyra M. Park T.J. Curran T. Crk and CrkL are required for cell transformation by v-fos and v-ras.Mol Carcinog. 2016; 55: 97-104Crossref PubMed Scopus (5) Google Scholar). Here we utilized the conditional knock-out system to examine whether endogenous Crk and CrkL are required for cell growth. Our findings clearly demonstrate that Crk and CrkL play essential, overlapping roles in fibroblast proliferation by enabling cells to progress from the G1 phase to the S phase. Our study also demonstrates that expression of any one protein among CrkI, CrkII, and CrkL at physiological levels is sufficient to secure cell proliferation. We used the Neon system (Life Technologies) to achieve rapid and efficient gene expression in growing fibroblasts by transduction of synthetic mRNA (synRNA). To reduce innate immune responses and increase ectopic protein expression, we synthesized mRNA using the modified ribonucleotides, pseudo-UTP, and methyl-CTP (23.Warren L. Manos P.D. Ahfeldt T. Loh Y.H. Li H. Lau F. Ebina W. Mandal P.K. Smith Z.D. Meissner A. Daley G.Q. Brack A.S. Collins J.J. Cowan C. Schlaeger T.M. Rossi D.J. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.Cell Stem Cell. 2010; 7: 618-630Abstract Full Text Full Text PDF PubMed Scopus (2078) Google Scholar, 24.Kuhn A.N. Beiβert T. Simon P. Vallazza B. Buck J. Davies B.P. Tureci O. Sahin U. mRNA as a versatile tool for exogenous protein expression.Curr. Gene Ther. 2012; 12: 347-361Crossref PubMed Scopus (46) Google Scholar). When fibroblasts immortalized by T antigen or the 3T3 protocol were transfected with synthetic green fluorescent protein mRNA (synGFP), both cell types in monolayer cultures showed evidence of green fluorescence at 1 day post transduction (DPT) (Fig. 1A). As cells proliferated, the intensity of fluorescence in individual cells gradually decreased (Fig. 1A). Similarly, the protein level of GFP in the cell population as measured by Western analysis displayed a similar trend (Fig. 1B). Time-lapse analysis of live fibroblasts transfected with synGFP showed strong GFP signals at 5 h post transduction, with the fluorescence reaching a maximum around 24 h post transduction, and thereafter, it gradually declined (Fig. 1, C and D). For quantification of the synRNA transfection efficiency, T antigen-immortalized fibroblasts were transfected with synGFP and fixed at different time points, and their nuclei were stained with DAPI. As shown in Fig. 1, E and F, 99 and 98% of fibroblasts were GFP-positive at 1 and 2 DPT, respectively. At 3 DPT, 69% of cells were positive for GFP. In contrast, only 5% of fibroblasts were GFP-positive at 2 and 3 DPT after GFP DNA transfection. The decrease in average GFP fluorescence intensity per cell over time is likely a consequence of the rapid proliferation of fibroblasts (Fig. 1G), thereby diluting the signal as the total intensities for all the fields of view (intensity × cell count) and the entire well (intensity x WST-1) stayed relatively constant over time (Fig. 1H). These observations suggest that electroporation of fibroblasts with synRNA is an effective method for rapid and efficient introduction of exogenous proteins into growing fibroblasts. To investigate the contribution of endogenous Crk and CrkL to cell growth, we immortalized fibroblasts from Crk/CrkL double-floxed mice using SV40 T antigen. We transfected the fibroblasts with synthetic Cre mRNA (synCre) to induce a rapid and efficient ablation of Crk and CrkL. As shown in Fig. 2A, staining of fibroblasts with crystal violet indicated substantial increases in cell number between 1 and 3 DPT for control and synGFP-transfected cells. In contrast, the cell number did not increase in cells transfected with synCre. Quantitative analyses of cell proliferation using WST-1 revealed similar exponential rates of cell proliferation in control and synGFP populations (slopes of 1.25 and 1.30, respectively) (Fig. 2, B–D). In contrast, synCre cells failed to proliferate (a slope of 0.05). Western analyses of cell lysates indicated that synCre induced efficient ablation of CrkI, CrkII, and CrkL. The steady state levels of Crk family proteins and other cellular proteins increased as control and synGFP cells recovered from trypsin-EDTA treatment and electroporation and continued to grow (Fig. 2E), probably due to increased cell-to-cell connections. It has been reported that expression and tyrosine phosphorylation of many cellular proteins, including focal adhesion proteins, increase at higher cell densities in Balb 3T3 fibroblasts (25.Batt D.B. Roberts T.M. Cell density modulates protein-tyrosine phosphorylation.J. Biol. Chem. 1998; 273: 3408-3414Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In contrast, in synCre cells, the levels of Crk family proteins declined after electroporation (Fig. 2, E–G). Tyrosine phosphorylation of p130Cas, a well known protein that associates with Crk family members, decreased as Crk family proteins disappeared from the cells (Fig. 2, E and H). Phosphorylation of Akt was also reduced in the absence of Crk and CrkL (Fig. 2, E and I). Although the level of β-actin showed a modest decrease, vinculin expression was not affected by the loss of Crk and CrkL (Fig. 2, J and K). The requirement of Crk and CrkL for cell proliferation and p130Cas phosphorylation was also observed in fibroblasts immortalized by the 3T3 protocol, which grew more slowly than T antigen-immortalized fibroblasts (data not shown). Our results indicate that endogenous Crk and CrkL are required for proliferation of immortalized fibroblasts. Staining of fibroblasts with Crk and CrkL antibodies suggests that Crk is expressed in the nucleus and cytoplasm with a higher expression in the nucleus, whereas CrkL is expressed in both the cytoplasm and the nucleus at similar levels (Fig. 3B, data not shown). When cells were transfected with synCre, the level of Crk substantially declined. On the other hand, the decrease in the CrkL level was not obvious in part because a polyclonal antibody for CrkL was used for the staining, although Western blot analysis clearly showed a substantial change in CrkL levels between the control and synCre cells (Fig. 2, E and G, versus Fig. 3B). Staining polymerized actin with phalloidin indicated that actin stress fiber formation was defective in the absence of Crk and CrkL. Cytoplasmic staining with a heat shock protein 90 antibody showed well developed cellular processes in control fibroblasts that were lost in the absence of Crk and CrkL, resulting in a more rounded appearance (Fig. 3, A and B). Without Crk and CrkL, cells formed tight clusters (Fig. 3A) that could be dissociated by treatment with trypsin-EDTA (data not shown), suggesting that they are not multinucleated cells. Because overexpression of Crk caused breakdown of adherens junctions and cell spreading as well as decreased levels of two key components of the adherens junction, E-cadherin and p120-catenin, in Madin-Darby canine kidney epithelial and non-small cell lung cancer cells (26.Lamorte L. Royal I. Naujokas M. Park M. Crk adapter proteins promote an epithelial-mesenchymal-like transition and are required for HGF-mediated cell spreading and breakdown of epithelial adherens junctions.Mol. Biol. Cell. 2002; 13: 1449-1461Crossref PubMed Scopus (108) Google Scholar, 27.Mortazavi F. Dubinett S. Rettig M. c-Crk proto-oncogene contributes to transcriptional repression of p120-catenin in non-small cell lung cancer cells.Clin. Exp. Metastasis. 2011; 28: 391-404Crossref PubMed Scopus (14) Google Scholar), we tested whether adherens junction formation changed in fibroblasts lacking both Crk and CrkL. As shown in Fig. 3B, staining of control fibroblasts with a p120-catenin antibody did not exhibit any junctional structure between neighboring cells. In contrast, staining of synCre-transfected fibroblasts with the same antibody showed well developed adherens junctions between the neighboring cells (yellow arrows in Fig. 3B). However, the E-cadherin antibodies we tested did not show such a clear formation of adherens junctions. On the other hand, a pan-cadherin antibody displayed cell-to-cell junctions in both control and synCre-transfected cells, and the junctions looked more concentrated in synCre cells (white arrows in Fig. 3B). It is unclear whether they were adherens junctions because the shapes (white arrows) appeared to be different from the adherens junctions stained with p120-catenin (yellow arrows). Our results are consistent with Crk-induced breakdown of adherens junctions reported by Lamorte et al. (26.Lamorte L. Royal I. Naujokas M. Park M. Crk adapter proteins promote an epithelial-mesenchymal-like transition and are required for HGF-mediated cell spreading and breakdown of epithelial adherens junctions.Mol. Biol. Cell. 2002; 13: 1449-1461Crossref PubMed Scopus (108) Google Scholar) and suggest that adherens junctions were formed between neighboring cells in the absence of Crk and CrkL, contributing to formation of cell clusters. Furthermore, both nuclear and cytoplasmic areas of the cells significantly decreased in the absence of Crk and CrkL (Fig. 3, C and D). In addition, the cytoplasmic circularity increased in the absence of Crk and CrkL (Fig. 3E), suggesting that cells shrank and became rounded. Next, we examined whether the altered cell structure and formation of adherens junctions affect cell motility. The scratch wound-healing assay revealed directional migration of wild-type fibroblasts to the wound site for 24 h with 44 ± 4% of cells located in the region of interest (ROI) (Fig. 4, A–C). In contrast, only 4 ± 4% of synCre-transfected fibroblasts migrated to the wound site. These results confirm our previous findings (21.Park T.J. Curran T. Essential roles of Crk and CrkL in fibroblast structure and motility.Oncogene. 2014; 33: 5121-5132Crossref PubMed Scopus (29) Google Scholar) and demonstrate that Crk and CrkL play essential roles in cell structure, formation of cell-to-cell junctions, and cell motility.FIGURE 4.Reduced cell motility in the absence of Crk and CrkL. T antigen-immortalized Crk/CrkL double-floxed fibroblasts were transfected without synRNA (None) or with synCre (0.2 μg in 10 μl cell suspension). At 2 DPT, a wound was created by scratching through the cell monolayer with a micropipette tip, and cells were allowed to migrate into the gap at the wound site for 24 h, fixed, and stained with a HSP90 antibody and DAPI to visualize the cytoplasm and nucleus. A, after fluorescence images were taken, a ROI of 2230 × 1000 μm as the original wound gap was drawn (red rectangles) using the Nikon NIS element program. Six separate experiments were done for each group, and representative images are shown. Scale bar: 500 μm. B, DAPI-stained objects were counted using the Nikon NIS element program for the whole image and for the ROI. Data are shown as the mean ± S.D. (bars) values. C, the percentage of cells that migrated to the ROI was calculated for each image. Data are shown as the mean ± S.D. (bars) values. ***, p < 0.001, compared with control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The highly efficient transduction of cells with synCre raised a potential concern of nonspecific toxicity of CRE. Previously, other groups associated high levels of CRE expression with inhibition of cell growth and cytopathic effects (28.Loonstra A. Vooijs M. Beverloo H.B. Allak B.A. van Drunen E. Kanaar R. Berns A. Jonkers J. Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 9209-9214Crossref PubMed Scopus (472) Google Scholar, 29.Pfeifer A. Brandon E.P. Kootstra N. Gage F.H. Verma I.M. Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 11450-11455Crossref PubMed Scopus (195) Google Scholar). Therefore, we conducted a dose-response analysis of synCre in wild-type and Crk/CrkL double-floxed fibroblasts. Transfection of wild-type fibroblasts with synCre up to 0.2 μg did not affect exponential growth of cells (Fig. 5, A and C). However, use of 0.66 μg and more of synCre reduced proliferation of wild-type cells in a dose-dependent manner. In contrast, as little as 0.066 μg of synCre inhibited proliferation of Crk/CrkL double-floxed cells, and at 0.2 μg there was essentially no growth (Fig. 5, B and C). Crystal violet staining of Crk/CrkL double-floxed cells showed that both inhibition of cell proliferation and morphological alteration occurred at a level of 0.2 μg synCre, indicating that both phenotypes are related (Fig. 5D). Examination of CrkI, CrkII, and CrkL indicated that all were effectively ablated using as little as 0.2 μg of synCre in double-floxed cells (Fig. 5, E–G). Furthermore, the changes in the growth rate and in the Crk family protein levels were closely correlated in Crk/CrkL double-floxed cells (Fig. 5, C versus F and G), suggesting that Crk family proteins play an essential role in cell proliferation. Phosphorylated forms of p130Cas and Akt were decreased to similar extents by 0.2 μg of synCre (Fig. 3, H and I). On the other hand, the decrease in β-actin levels induced by 2 μg synCre in wild-type cells seems to reflect a nonspecific effect of synCre. These results suggest that 0.2 μg is the optimal amount of synCre to investigate the effects of Crk/CrkL ablation without confounding nonspecific effects of high levels of CRE. Therefore, we chose to use 0.2 μg of synCre for further analyses. Because transfection of Crk/CrkL double-floxed cells with synCre ablated both Crk and CrkL, it was unclear which of these proteins was actually required for cell proliferation. To ablate Crk or CrkL alone, we derived immortalized fibroblasts from Crk or CrkL floxed mice and transfected them with synCre. As shown in Fig. 6, A and B, loss of either Crk or CrkL only slightly reduced cell proliferation (10.8% and 4.9% inhibition for single-floxed cells compared with 92.4% for double-floxed cells). Furthermore, loss of either Crk or CrkL did not result in any detectable morphological alterations (Fig. 6C). Western analyses verified the loss of Crk or CrkL alone upon synCre transfection in single-floxed cells (Fig. 6, D and E). It is noteworthy that phosphorylated p130Cas decreased only when cells lost Crk (or Crk plus CrkL). The result suggests that Crk predominantly mediates phosphorylation of p130Cas. Phosphorylation of Akt decreased in the absence of both Crk and CrkL, but it did not decrease significantly when cells lacked either Crk or CrkL, suggesting that both Crk and CrkL contribute to Akt phosphorylation, with either sufficient for the phosphorylation to occur. The results from single-floxed fibroblasts demonstrate that Crk and CrkL play overlapping functions in cell proliferation and that ablation of one does not cause obvious defects.
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