Pdx-1 Activates Islet α- and β-Cell Proliferation via a Mechanism Regulated by Transient Receptor Potential Cation Channels 3 and 6 and Extracellular Signal-Regulated Kinases 1 and 2

Abstract

The homeodomain transcription factor Pdx-1 has important roles in pancreatic development and ␤-cell function and survival. In the present study, we demonstrate that adenovirus-mediated overexpression of Pdx-1 in rat or human islets also stimulates cell replication. Moreover, cooverexpression of Pdx-1 with another homeodomain transcription factor, Nkx6.1, has an additive effect on proliferation compared to either factor alone, implying discrete activating mechanisms. Consistent with this, Nkx6.1 stimulates mainly ␤-cell proliferation, whereas Pdx-1 stimulates both ␣-and ␤-cell proliferation. Furthermore, cyclins D1/D2 are upregulated by Pdx-1 but not by Nkx6.1, and inhibition of cdk4 blocks Pdx-1-stimulated but not Nkx6.1-stimulated islet cell proliferation. Genes regulated by Pdx-1 but not Nkx6.1 were identified by microarray analysis. Two members of the transient receptor potential cation (TRPC) channel family, TRPC3 and TRPC6, are upregulated by Pdx-1 overexpression, and small interfering RNA (siRNA)-mediated knockdown of TRPC3/6 or TRPC6 alone inhibits Pdx-1-induced but not Nkx6.1-induced islet cell proliferation. Pdx-1 also stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, an effect partially blocked by knockdown of TRPC3/6, and blockade of ERK1/2 activation with a MEK1/2 inhibitor partially impairs Pdx-1-stimulated proliferation. These studies define a pathway by which overexpression of Pdx-1 activates islet cell proliferation that is distinct from and additive to a pathway activated by Nkx6.1.

A Pdx-1-Regulated Soluble Factor Activates Rat and Human Islet Cell Proliferation

“…For gene overexpression studies, cytomegalovirus (CMV) promoter-driven recombinant adenoviruses containing mouse Pdx-1 (AdCMV-mPdx-1), bacterial ␤-galactosidase (AdCMV-␤-Gal), or green fluorescent protein (GFP; AdCMV-GFP) were constructed and used as described previously (8,9,13). We constructed CMV promoter-driven adenoviruses containing human Pdx-1 (hPdx-1), rat Noggin, rat Bmp3, rat Fstl5, and rat Inhbb by cloning the cDNA constructs into a pAdTrack shuttle vector and using the Ad-Easy system to generate the recombinant adenoviruses, as previously described (14).…”

“…Our laboratory has demonstrated that Pdx-1 and Nkx6.1, two homeobox domain transcription factors well known for key roles in islet ␤-cell development, activate proliferation when overexpressed in rat or human islets while maintaining and enhancing GSIS, respectively (7)(8)(9)(10). Importantly, the increase in proliferation induced by either factor has little to no impact on ␥-H2AX expression, suggesting that these factors engage "safe" pathways of islet cell replication.…”

“…These positive features of Nkx6.1-and Pdx-1-mediated islet cell replication have led us to explore the pathways by which they engage the core cell cycle machinery (7)(8)(9)(10). In our previous studies using adenovirus vectors containing the constitutive cytomegalovirus (CMV) promoter to drive transgene expression, we found that Nkx6.1 overexpression in rat islets causes almost exclusive proliferation of ␤ cells, whereas Pdx-1 overexpression activates both ␤-and ␣-cell replication (7,9,10).…”

“…In our previous studies using adenovirus vectors containing the constitutive cytomegalovirus (CMV) promoter to drive transgene expression, we found that Nkx6.1 overexpression in rat islets causes almost exclusive proliferation of ␤ cells, whereas Pdx-1 overexpression activates both ␤-and ␣-cell replication (7,9,10). Here, we sought to understand the molecular pathway by which Pdx-1 overexpression activates both ␣-and ␤-cell replication.…”

The Making of Pancreatic β Cells: Advances and Apprehensions

“…[ [81][82][83][84][85][86][87][88][89] Ngn-3 Promotes ectopic development of beta and delta cells.Associated with beta cell neogenesis and exocrine to endocrinal conversion.…”