Knockdown Of SHP2 Expression Research Articles

SHP2 regulates the development of intestinal epithelium by modifying OSTERIX+ crypt stem cell self-renewal and proliferation.

The protein tyrosine phosphatase SHP2, encoded by PTPN11, is ubiquitously expressed and essential for the development and/or maintenance of multiple tissues and organs. SHP2 is involved in gastrointestinal (GI) epithelium development and homeostasis, but the underlying mechanisms remain elusive. While studying SHP2's role in skeletal development, we made osteoblast-specific SHP2 deficient mice using Osterix (Osx)-Cre as a driver to excise Ptpn11 floxed alleles. Phenotypic characterization of these SHP2 mutants unexpectedly revealed a critical role of SHP2 in GI biology. Mice lacking SHP2 in Osx+ cells developed a fatal GI pathology with dramatic villus hypoplasia. OSTERIX, an OB-specific zinc finger-containing transcription factor is for the first time found to be expressed in GI crypt cells, and SHP2 expression in the crypt Osx+ cells is critical for self-renewal and proliferation. Further, immunostaining revealed the colocalization of OSTERIX with OLFM4 and LGR5, two bona fide GI stem cell markers, at the crypt cells. Furthermore, OSTERIX expression is found to be associated with GI malignancies. Knockdown of SHP2 expression had no apparent influence on the relative numbers of enterocytes, goblet cells or Paneth cells. Given SHP2's key regulatory role in OB differentiation, our studies suggest that OSTERIX and SHP2 are indispensable for gut homeostasis, analogous to SOX9's dual role as a master regulator of cartilage and an important regulator of crypt stem cell biology. Our findings also provide a foundation for new avenues of inquiry into GI stem cell biology and of OSTERIX's therapeutic and diagnostic potential.

SHP‐2 phosphatase promotes cervical cancer cell proliferation through inhibiting interferon‐β production

The aim of this study was to explore the effect of Src-homology-2-domain-containing protein, tyrosine phosphatase 2 (SHP-2) on the proliferation of cervical cancer cells. A total of 45 patients with cervical cancer (stage I-III), 32 with cervical intraepithelial neoplasia and 20 healthy subjects were consecutively recruited. The levels of SHP-2 and interferon (IFN)-β expression in cervical tissues were characterized by immunohistochemistry and statistically analyzed by logistic regression. Following knockdown of SHP-2 expression by a siRNA or pre-treatment with a specific peptide, the effect of SHP-2 expression in THP-1 cells on the growth and survival of SiHa cells and on IFN-β production was determined by co-culture assays, 3-(4,5)-dimethylthiazol (-z-y 1)-3,5-diphenyltetrazolium bromide, and enzyme immunosorbent assay. The levels of SHP-2 expression in cervical cancer tissues were significantly higher than that in cervical intraepithelial neoplasia and uterine myoma tissues (P<0.05, respectively), and negatively correlated with the levels of IFN-β expression in these tissues (R=-0.582, P<0.05). Knockdown of SHP-2 expression with SHP-2 siRNA or treatment with the SHP-2-specific blocking peptide in THP-1 cells significantly increased the production of IFN-β (P<0.05, respectively) and inhibited the proliferation of SiHa cells in a co-culture system of THP-1 and SiHa cells (P<0.05, respectively). SHP-2 phosphatase promotes cervical cancer cell proliferation through inhibiting IFN-β production.

Abstract 4097: Protein tyrosine phosphatase SHP-2 regulates human pancreatic cancer cell biology on collagen I

Abstract With a five year survival rate of less than 5%, pancreatic cancer stands as one of the deadliest human malignancies. There is a great need for novel therapeutic strategies for this illness, and improving our understanding of pancreatic cancer biology will be a key step in developing these therapies. The purpose of these studies is to investigate the regulation and function of the Src Homology 2 (SH2) domain containing protein tyrosine phosphatase SHP-2 in human pancreatic cancer cells and to determine its potential value as a therapeutic target for this disease. In other cellular systems, SHP-2 is crucial for integrin and receptor tyrosine kinase-mediated activation of diverse signaling pathways, regulating many of the cellular events necessary for oncogenesis, including cell division, motility, adhesion, and survival. We hypothesized that inhibition of SHP-2 function would negatively impact integrin-mediated cell signaling and biology in response to the extracellular matrix protein collagen I, an abundant component of pancreatic tumor stroma in vivo. Initial results demonstrate that SHP-2 expression and tyrosine phosphorylation/activity are elevated in pancreatic cancer cells, relative to immortalized normal human pancreatic cells. Phosphorylation of SHP-2 is further elevated in the presence of collagen I. SHP-2 tyrosine phosphorylation in pancreatic cancer cells occurrs in a Src family kinase-dependent fashion. Initial results using miRNA-mediated knock-down of SHP-2 expression indicate the importance of SHP-2 for the ability of pancreatic cancer cells to proliferate, migrate and heal cell culture wounds on collagen I. Additionally, SHP-2 knockdown cells grown on collagen I demonstate altered tyrosine phosphorylation patterns of several signal transduction proteins, including focal adhesion kinase (FAK), in comparison to controls. These data suggest, for the first time, an importance for SHP-2 in pancreatic cancer cell biology and the potential value of SHP-2 as a therapeutic target for this disease. Ongoing and future experiments will compare the effects of SHP-2 inhibition on pancreatic tumor growth in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4097.

374. Knockdown of Shp-2 Expression by siRNA in Mouse Embryonic Stem Cell Derived EB Cells Affects Their Hematopoietic Development

Shp-2 is a member of a small subfamily of cytoplasmic Src homology 2 (SH2) domain-containing protein tyrosine phosphatases. To address the question of whether Shp-2 is required for normal hemangioblast, primitive, and definitive hematopoietic development, we used Shp-2 siRNA to knockdown Shp-2 gene expression in embryonic stem (ES) derived embryoid body (EB) cells. We found that a pool of (but not single siRNA) Shp-2 siRNA at a concentration of 50 nM/ml was efficient to knockdown Shp-2 expression in EB cells. The density of Shp-2 protein in western blot assay was decreased about 10 fold in Shp-2 siRNA treated cells compared with cells treated with a scrambled control Shp-2 siRNA. The frequency of growth factor-dependent hemangioblast formation were decreased 5 fold following cell treatment with Shp-2 siRNA (112.6 |[plusmn]| 11.7 vs 31 |[plusmn]| 1), but not by scrambled Shp-2 siRNA (112.6 |[plusmn]| 11.7 vs 114 |[plusmn]| 12). Basic fibroblast growth factor (bFGF) promotes hemangioblast development. As Shp-2 is involved in the upstream signaling of bFGF receptor activation, we then asked whether Shp-2 was involved in this bFGF mediated hemangioblast effect. Addition of bFGF at doses of 10 and 20 ng/ml significantly increased (121.6 |[plusmn]| 4.7 vs 232.3 |[plusmn]| 10) and (121.6 |[plusmn]| 4.7 vs 281.3 |[plusmn]| 4.1) (compared with control siRNA treated cells) the frequency of hemangioblast formation. However, knockdown of Shp-2 gene expression by siRNA blocked the bFGF effect on hemangioblast development. In primitive hematopoietic assays, knockdown of Shp-2 expression in day-6 EB cells resulted in a (350 |[plusmn]| 12.5 vs 173 |[plusmn]| 16.3) 2 fold decrease in EPO-dependent Ery-P colonies, a (25 |[plusmn]| 2.3 vs 7.2 |[plusmn]| 1.8) 3 fold decrease in Ery-D colonies and a (23 |[plusmn]| 1.8 vs 4.1 |[plusmn]| 0.6) 5 fold decrease in GM-CSF-dependent CFU-GM colonies. Altogether, these data demonstrate that Shp-2 is required in hemangioblast, primitive, and definitive progenitor hematopoietic development. Shp-2 is also involved in bFGF regulation of hemangioblast development. We conclude that Shp-2 plays a critical role in hemangioblast, primitive, and definitive hematopoiesis.

Helicobacter pylori CagA Induces Ras-independent Morphogenetic Response through SHP-2 Recruitment and Activation

The CagA protein of Helicobacter pylori, which is injected from the bacteria into bacteria-attached gastric epithelial cells, is associated with gastric carcinoma. CagA is tyrosine-phosphorylated by Src family kinases, binds the SH2 domain-containing SHP-2 phosphatase in a tyrosine phosphorylation-dependent manner, and deregulates its enzymatic activity. We established AGS human gastric epithelial cells that inducibly express wild-type or a phosphorylation-resistant CagA, in which tyrosine residues constituting the EPIYA motifs were substituted with alanines. Upon induction, wild-type CagA, but not the mutant CagA, elicited strong elongation of cell shape, termed the "hummingbird" phenotype. Time-lapse video microscopic analysis revealed that the CagA-expressing cells exhibited a marked increase in cell motility with successive rounds of elongation-contraction processes. Inhibition of CagA phosphorylation by an Src kinase inhibitor, PP2, or knockdown of SHP-2 expression by small interference RNA (siRNA) abolished the CagA-mediated hummingbird phenotype. The morphogenetic activity of CagA also required Erk MAPK but was independent of Ras or Grb2. In AGS cells, CagA prolonged duration of Erk activation in response to serum stimulation. Conversely, inhibition of SHP-2 expression by siRNA abolished the sustained Erk activation. Thus, SHP-2 acts as a positive regulator of Erk activity in AGS cells. These results indicate that SHP-2 is involved in the Ras-independent modification of Erk signals that is necessary for the morphogenetic activity of CagA. Our work therefore suggests a key role of SHP-2 in the pathological activity of H. pylori virulence factor CagA.