Abstract
The homeodomain transcription factor Pdx-1 has important roles in pancreatic development and β-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 β-cell proliferation, whereas Pdx-1 stimulates both α- and β-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.
SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00469-13.
ACKNOWLEDGMENTS
This work was supported by a grant from the National Institutes of Health β-Cell Biology Consortium (BCBC) (U01 DK-089538) to H.E.H. and C.B.N., Juvenile Diabetes Research Foundation (JDRF) grants 17-2011-15 (to C.B.N.) and 17-2011-614 (to H.E.H.), and a JDRF postdoctoral fellowship to H.L.H. (3-2009-561).
We thank Samuel Stephens and Jeffery Tessem for helpful advice and discussion, as well as Danhong Lu, Helena Winfield, Lisa Poppe, and Paul Anderson for expert technical assistance. We thank the Duke Microarray Core facility (a Duke National Cancer Institute and a Duke Institute for Genome Sciences and Policy shared resource facility) for their assistance in generating the microarray data reported in the manuscript.