Pro-inflammatory
cytokines are implicated as key mediators of beta-cell death during type 1
diabetes and islet transplantation. This may involve up and/or
down regulation of gene transcription. In order to uncover new
transcription factors (TFs) involved in this process, siRNA’s of ~600 human TFs
were transfected into dispersed primary human pancreatic islets. 48h post
transfection the islets were treated with a combination of TNFa, IL-1b, and IFNg for 24h followed by measurement of caspase-3/7 activity as an
apoptosis marker. This
screen identified a number of novel pro- and anti-apoptotic TFs that had not
been previously associated with pancreatic beta cell death. One such TF was the
Hairy and Enhancer of Split 4 (HES4), a human TF whose mode of action is poorly
understood. The Hes gene family
encodes transcriptional regulators of the bHLH class that mainly act as
repressors. Silencing of HES4 expression in dispersed
human islets from 5 donors significantly increased cytokine-induced caspase 3/7
activity. Similar results were obtained with human liver hepatocellular
carcinoma cell line (HepG2). More specifically, INFg, in
combination with either TNFa or
IL-1b, induced this
effect. Of note, neither Hes1 nor Hes3, the
other members of the Hes subfamily, had similar effects. At
the molecular level, silencing of Hes4 mRNA in HepG2 resulted in increased expression
of the cyclin-dependent kinase inhibitors p27(Kip1) and
p21(Cip1). Interestingly, whereas iNOS expression was
significantly increased it was not crucial for the cytokine-induced cell death
in either human islets or HepG2 cells. Of note, treatment with cytokines
transiently increased Hes4 expression in both. Taken together, our results
implicate Hes4 as a novel human TF involved in the preservation of cell viability
and in anti inflammatory pathways. A negative feedback control is proposed
whereby cytokines induce Hes4 expression which then inhibits their function. As
such, it might be a novel target for therapeutic intervention in cases
of cancer.
