Expression of c-Kit receptor tyrosine kinase and effect on β-cell development in the human fetal pancreas

Abstract

The receptor, c-Kit, and its ligand, stem cell factor (SCF), are critical for hematopoietic stem cell differentiation and have been implicated in the development, function, and survival of rodent islets. Previously, we reported that exogenous SCF treatments of cultured human fetal (14-16 wk fetal age) islet-epithelial clusters enhanced islet cell differentiation and proliferation (Li J, Goodyer CG, Fellows F, Wang R. Int J Biochem Cell Biol 38: 961-972, 2006). In the present study, we examined the expression pattern of c-Kit in early to midgestation human fetal pancreata and the relevance of c-Kit receptor tyrosine kinase for insulin gene expression and beta-cell survival. c-Kit is expressed in the intact pancreas in a cell-specific manner, with a significant decrease in immunoreactivity in the duct regions from 8 to 21 wk fetal age, paralleled by a significant increase in expression within endocrine regions. These c-Kit-positive cells are highly proliferative and show frequent coexpression with insulin and glucagon. Treatment of islet-epithelial clusters with anti-ACK45 antibody stimulates c-Kit phosphorylation paralleled by a significant increase in PDX-1 and insulin expression, increased cell proliferation, and reduced beta-cell death. In contrast, transient transfection with c-Kit siRNA results in a three- to fourfold decrease in c-Kit, PDX-1, and insulin expression and decreased cell proliferation. This study describes important changes in the distribution and dynamics of c-Kit-expressing cells during human fetal pancreatic neogenesis, suggesting that c-Kit may be a marker for human pancreatic islet progenitor cells. Functional analysis of the c-Kit receptor tyrosine kinase provides evidence that phosphorylation of c-Kit receptor may be involved in mediating early beta-cell differentiation and survival.

A survival Kit for pancreatic beta cells: stem cell factor and c-Kit receptor tyrosine kinase

“…Furthermore, W and Sl mutant mice display increased germ cell apoptosis, with a corresponding degree of sterility [41]. More importantly, accumulating evidence suggests that c-Kit expression is also linked to the development of the endocrine pancreas, as well as to beta cell survival and function [11,12,[42][43][44][45][46][47][48][49][50][51][52][53].…”

“…The developing human pancreas and multiple human pancreatic cancer cell lines express both c-Kit and SCF (Table 1) [11,49,53,56]. c-Kit + cells were detected at the earliest stage of human pancreatic development, as single endocrine cells differentiated from ducts [49].…”

Effect of forkhead box O1 (FOXO1) on beta cell development in the human fetal pancreas

“…FBS and transiently transfected with 60 nmol/l human FOXO1 siRNA (sc-35382) or control siRNA (sc-37007, proprietary sequence) commercially obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), using an siRNA transfection kit [19,20]. A pool of three sequences for human FOXO1 siRNA (Santa Cruz Biotechnology) was used (Electronic supplementary material [ESM] Table 1).…”

“…Islet epithelial cell clusters were harvested at 24 and 48 h following transfection. Transfection efficiency was monitored using fluorescein-conjugated control siRNA (Santa Cruz Biotechnology) and quantitative RT-PCR (qRT-PCR) analysis for FOXO1 mRNA, with approximately 60% of the islet epithelial cluster cells being transfected, as described previously [19][20][21][22].…”

“…Densitometric quantification of bands at subsaturation levels was performed using Syngenetool gel analysis software (Syngene, Cambridge, UK) and normalised to appropriate loading controls, which included the total signalling protein content or housekeeping proteins (calnexin, β-actin). Data are provided as the relative abundance of phosphorylated proteins to total protein levels or protein levels to the loading control (calnexin, β-actin) [20,21].…”