GRB2 serves as a viable target against skin fibrosis in systemic sclerosis by regulating endothelial cell apoptosis.

BACKGROUND: Systemic Sclerosis (SSc) is an autoimmune disease characterized by vascular and immune system dysfunction, along with tissue fibrosis. Our previous study found GRB2 was downregulated by salvianolic acid B, a small molecule drug that attenuated skin fibrosis of SSc. OBJECTIVES: Here we aim to investigate the role of GRB2 in SSc. METHODS: The microarray data of SSc skin biopsies in Caucasians were obtained from the Gene Expression Omnibus (GEO) database. The expression of GRB2 was further detected in Chinese SSc and healthy controls. Bleomycin (BLM)-induced skin fibrosis mice were used to explore how GRB2 downregulation affected fibrosis. The apoptosis of EA.hy926 endothelial cells was induced by H2O2 and apoptosis ratio was measured by flow cytometric. Transcriptome and phosphoproteomic analyses were performed to explore the regulated pathway. RESULTS: The expression of GRB2 was significantly enhanced in SSc patient skin, 1.51-fold in Caucasians and 1.40-fold in Chinese. Double immunofluorescence staining showed the endothelial cells of SSc patient's skin highly expressed GRB2. The in vivo study revealed that GRB2 knockdown alleviated skin fibrosis and apoptosis of endothelial cells in BLM mouse skin. The in vitro study showed that GRB2 downregulation inhibited the apoptosis of EA.hy926 and protected them from H2O2-induced hyperpermeability. Moreover, transcriptome and phosphoproteomic analysis suggested the focal adhesion pathway was enriched in GRB2 siRNA transfected endothelial cells. CONCLUSIONS: Our results demonstrated GRB2 highly expressed in endothelial cells of SSc skin, and inhibiting GRB2 could effectively attenuate BLM-induced skin fibrosis and endothelial cell apoptosis. GRB2 is expected to be a new therapeutic target for SSc.

Effects of RING-SH2Grb², a chimeric protein containing the E3 ligase domain of Cbl, on the EGFR pathway.

The E3 ubiquitin-protein ligase Casitas B-lineage lymphoma protein (Cbl) negatively regulates epidermal growth factor receptor (EGFR) signaling pathway in many organisms, and has crucial roles in cell growth, development and human pathologies, including lung cancers. RING-SH2Grb² a chimeric protein of 215 amino acids containing the RING domain of Cbl that provides E3 ligase activity, and the SH2 domain of Grb2 that serves as an adaptor for EGFR. In this study, we demonstrated that RING-SH2Grb² could promote the ubiquitinylation and degradation of EGFR in a human non-small cell lung carcinoma cell line H1299. Moreover, we discovered that the RING-SH2Grb² chimera promoted the internalization of ligand-bound EGFR, inhibited the growth of H1299 cells, and significantly suppressed tumor growth in a xenograft mouse model. In summary, our results revealed a potential new cancer therapeutic approach for non-small cell lung cancer.

Caenorhabditis elegans fibroblast growth factor receptor signaling can occur independently of the multi-substrate adaptor FRS2.

The components of receptor tyrosine kinase signaling complexes help to define the specificity of the effects of their activation. The Caenorhabditis elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2-binding motifs present in the CTD (Y(1009) and Y(1087)), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. SEM-5, but not the SEM-5 binding sites located in the CTD, is required for the fluid homeostasis function of EGL-15, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multi-substrate adaptor protein FRS2 serves to link vertebrate FGFRs to Grb2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/MAPK pathway for oocyte maturation but is not required for EGL-15 function. Thus, unlike the vertebrate FGFRs, which require the multi-substrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly.

A potential role for calcium / calmodulin-dependent protein kinase-related peptide in neuronal apoptosis: in vivo and in vitro evidence.

Previously, we have established that a product of the doublecortin-like kinase (DCLK) gene, DCLK-short, is cleaved by caspases during serum deprivation. Subsequently, the N-terminal cleavage product of DCLK-short facilitates apoptosis in the neuroblastoma cell line NG108. As this N-terminal cleavage product is highly homologous to calcium/calmodulin-dependent protein kinase-related peptide (CARP), another DCLK gene splice variant, we aimed to determine the possible apoptotic properties of CARP in vivo and in vitro. We report highly specific CARP expression in apoptotic granule cells in the rat dentate gyrus after adrenalectomy relative to healthy granule cells. CARP is significantly upregulated in the suprapyramidal blade of the dentate gyrus, with varying levels of upregulation, depending on the extent of adrenalectomy-induced apoptosis. Similar to the caspase-cleaved N-terminus of DCLK-short, CARP overexpression itself facilitated apoptosis in serum-deprived NG108 cells. Furthermore, CARP facilitated polymerization of tubulin in vitro and was capable of interacting with growth factor receptor-bound protein 2, an intracellular protein involved in vesicle trafficking. Together, our data demonstrate a facilitating role for CARP in the apoptotic process in granule cell populations sensitive to adrenalectomy, and suggest that this proapoptotic effect is mediated by increasing the stability of the microtubule cytoskeleton.

A novel avian homologue of CD72, chB1r, down modulates BCR-mediated activation signals.

The avian B cell differentiation antigen chB1 is a C-type lectin membrane protein most homologous to mammalian CD72. Here, we report a new chB1-related gene, chB1r, that is located 18 kb away the chB1 gene. The cytoplasmic domain of chB1r protein contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs: ITIM1 and 2), which are identical to those found in CD72, whereas chB1 lacks the second ITIM2. Although chB1 expression is restricted to the bursa and an immature B cell line, chB1r is highly expressed in the bursa, spleen and both immature and mature B cell lines, a pattern that parallels CD72 expression. SHP-1 and Grb2 interact with phosphorylated tyrosine residues within chB1r ITIM1 and ITIM2, respectively. By contrast, ITIM1 of chB1 does not interact with SHP-1. Functional characterization using chB1r/chB1 double-deficient DT40 B cells demonstrated that ITIM1 in chB1r transduces a negative signal for BCR-mediated nuclear factor of activated T cells (NF-AT) activation and that ITIM2 attenuates this negative signal. This study has established chB1r as the genuine avian homologue of mammalian CD72, and revealed an opposing role for the two ITIMs through binding with SHP-1 and Grb2 for regulation of BCR-mediated NF-AT activation.