Down-regulation of pancreatic and duodenal homeobox-1 by somatostatin receptor subtype 5: a novel mechanism for inhibition of cellular proliferation and insulin secretion by somatostatin

Somatostatin (SST) is a regulatory peptide and acts as an endogenous inhibitory regulator of the secretory and proliferative responses of target cells. SST’s actions are mediated by a family of seven transmembrane domain G protein-coupled receptors that comprise five distinct subtypes (SSTR1-5). SSTR5 is one of the major SSTRs in the islets of Langerhans. Homeodomain-containing transcription factor pancreatic and duodenal homeobox-1 (PDX-1) is essential for pancreatic development, β cell differentiation, maintenance of normal β cell functions in adults and tumorigenesis. Recent studies show that SSTR5 acts as a negative regulator for PDX-1 expression and that SSTR5 mediates somatostatin’s inhibitory effect on cell proliferation and insulin expression/excretion through down-regulating PDX-1 expression. SSTR5 exerts its inhibitory effect on PDX-1 expression at both the transcriptional level by down-regulating PDX-1 mRNA and the post-translational level by enhancing PDX-1 ubiquitination. Identification of PDX-1 as a transcriptional target for SSTR5 may help in guiding the choice of therapeutic cancer treatments.

SST's effects on cellular functions are at least in part mediated by its ability to regulate transcription factors. For example, octreotide, a somatostatin analog, inhibits mRNA expression of transcription factor c-fos and AP-1 binding activity stimulated by dibutyryl adenosine 3 ,5 -cyclic monophosphate, serum and phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (Todisco et al., 1994(Todisco et al., , 1995. In this mini review, we summarize recent findings that PDX-1 acts as another transcription factor that is subject to the inhibitory regulation by SSTR5-mediated somatostatin signaling.

NEGATIVE REGULATION OF PDX-1 BY SSTR5 SSTR5 DOWN-REGULATES PDX-1
PDX-1 is subject to positive regulation by glucose (21), glucagon-like peptide 1 (GLP-1) (Buteau et al., 1999;Wang et al., 1999;Movassat et al., 2002;Li et al., 2005) and palmitic acid (Arantes et al., 2006). PDX-1 also is negatively regulated by DNA damage (Lebrun et al., 2005), oxidative stress (Boucher et al., 2006), and advanced glycation end-products (AGEs) (Puddu et al., 2010) under different physiological conditions. RPL-1980 is a SSTR5 specific agonist which proves that SSTR5 is the SSTR subtype responsible for inhibiting glucose-stimulated insulin secretion (Fagan et al., 1998;Zhou et al., 2011c). Recent studies show that RPL-1980 abolishes GLP-1-stimulated PDX-1 expression in mouse insulinoma β-TC6 cells , indicating that SSTR5-mediated somatostatin signaling is a potential novel negative regulator for PDX-1 expression. Further biochemical and genetic studies confirm the negative regulation of PDX-1 expression by SSTR5 . The evidence includes: (1) increased expression of SSTR5 inhibits PDX-1 expression likely due to increased PDX-1 ubiquitination; and (2) knockdown of SSTR5 by SSTR5 shRNA in β-TC6 cells results in increased PDX-1 expression and enhanced insulin secretion in response to a high concentration of glucose. These findings are consistent with the fact that SSTR5 mediates the inhibitory effect of somatostatin on insulin secretion (Fagan et al., 1998;Tirone et al., 2003). A SSTR5 knockdown-induced increase of PDX-1 expression in mouse insulinoma MIN6 cells is accompanied by elevated expression of cyclin E and cdk4 and decreased expression of p21, p27, and p53, supporting previous studies showing that SSTR5 inhibits cell proliferation (Fagan et al., 1998;Feanny et al., 2008) and promotes apoptosis (Qiu et al., 2006). This also is consistent with a previous study showing that SSTR5 contributes to the induction of cyclin-dependent kinase inhibitor p27 (Grant et al., 2008); and (3) genetic ablation of SSTR5 results in increased expression of PDX-1, which is accompanied by increased expression of insulin and proliferating cell nuclear antigen (PCNA) in sstr5 −/− islets. Also consistent is the finding that all three SSTR5 knockout mice (general SSTR5 −/− , β cell-specific SSTR5 −/− and SSTR1/5 −/− , SSTR1 and SSTR5 double knockout) develop islet hyperplasia, increased numbers of islets and islet neogenesis compared with littermate wild type controls (Wang et al., , 2005a. Given the essential role of PDX-1 in insulin expression (Ashizawa et al., 2004;Kaneto et al., 2007) and cell proliferation (Feanny et al., 2008), these studies support a new concept that SSTR5 may mediate the inhibitory effect of somatostatin on insulin expression and secretion and cell proliferation through a mechanism involving inhibiting PDX-1 expression.

PERSPECTIVE
Identification of PDX-1 as a novel transcriptional target for SSTR5-mediated somatostatin signaling greatly enhances our understanding of the cellular actions of somatostatin. SSTR5 has been shown to regulate a specific set of genes linked to cell proliferation, apoptosis, angiogenesis, immunity and tumorigenesis (Patel et al., 2009). PDX-1 also regulates a group of genes related to cell proliferation, apoptosis, invasion and angiogenesis (Liu et al., 2011). Given the negative regulation of PDX-1 by SSTR5 , it is likely that somatostatin exerts its cellular actions through regulating PDX-1-targeted genes with SSTR5. In addition to SSTR5, SSTs also exert their actions through SSTR1, 2, 3, and/or 4 under different cellular context. Therefore, it will be important to further determine if other SSTR-mediated cellular functions of somatostatin also involve PDX-1.
Little is known about the molecular mechanism by which SST/SSTR5 signaling down-regulates PDX-1. The observations that SSTR5 enhances, while SSTR5 P335L inhibits, PDX-1 ubiquitination  suggest that regulation of PDX-1 ubiquitination is a key participant in the pathways that connects SSTR5 with PDX-1. It has been shown that PDX-1 ubiquitination is subject to regulation by phosphorylation. DNAdependent protein kinase (DNA-PK) phosphorylates PDX-1 on Thr 11 and drives PDX-1 degradation by proteasome in response to DNA damage stimulation (Lebrun et al., 2005). Oxidative stress (H 2 O 2 )-stimulated, GSK-3-mediated phosphorylation of Ser 61 and Ser 66 also promotes PDX-1 proteasomal degradation (Boucher et al., 2006). ERK MAP kinases have kinase activity toward PDX-1 (Khoo et al., 2003). It has been shown that SSTmediated growth inhibition of human pituitary adenoma and GH3 cells is associated with the down-regulation of pERK and upregulation of p27 (Hubina et al., 2006). In addition, somatostatin exerts its anti-migratory and anti-invasive function through inhibition of ERK 1/2 signaling via the inhibition of small G protein Rac in SHSY5Y cells (Pola et al., 2003). Thus, ERK MAP kinase is one of downstream signaling pathways for somatostatin. It is, thus, reasonable to speculate that somatostatin/SSTR5 signaling may inhibit EGF-stimulated ERK kinase activation, leading to down-regulation of PDX-1 expression through a mechanism involved in the regulation of PDX-1 ubiquitination and stability (Figure 1). PDX-1 is a transcription factor for PDX-1 itself (Ashizawa et al., 2004). Thus, it is possible that downregulation of PDX-1 mRNA by SSTR5 is due to the downregulation of PDX-1 expression by SSTR5, which, in turn, results in decreased PDX-1 transcriptional activity. Alternatively, SSTR5 may directly down-regulate PDX-1 mRNA stability, leading to decreased PDX-1 mRNA expression.
SST is an endogenous antiproliferative agent and, thus, a promising antitumor agent (Florio, 2008;Pyronnet et al., 2008;Bodei et al., 2009;Modlin et al., 2009). SSTR5 and other SSTRs are widely and variably expressed in a variety of tumors such as gastroenteropancreatic tumors, pituitary tumors, and carcinoid tumors. Thus, these receptors represent the molecular basis for the clinical use of somatostatin analogs in the treatment of endocrine tumors. However, it has been challenging that only about 50% of patients with insulinoma are responsive to the treatment of somatostatin analog and lack long-term responsiveness. It could be due to the lack of expression of the target SSTRs on the tumor surface, since 10-50% of these tumors do not express SSTRs. Genetic variation(s) within the receptors that affect their cellular functions such as SSTR5 P335L, a hypofunctional SNP (Zhou et al., 2011c), may also contribute to the non-responsiveness to the treatment of somatostatin analogs. Silencing PDX-1 efficiently inhibits tumor cell proliferation in vitro and tumor growth in vivo (Liu et al., , 2011, showing that PDX-1 is a promising therapeutic target in cancer treatment. Thus, it is reasonable to speculate that a combined SSTR5-based tumor therapeutic approach including both somatostatin analog and other inhibiting technology of PDX-1 [for example, RNA interference control of PDX-1 ] would help improve the traditional treatment with somatostatin analogs. In patients with wild-type SSTR5, knockdown PDX-1 by biPDX-1 shRNA would enhance the therapeutic effect of somatostatin analog. In the case that genetic variations exist such as SSTR5 P335L, efficient knockdown PDX-1 by shRNA would help bypass the hypofunctional effect of SSTR5 P335L. Thus, identification of PDX-1 as a transcriptional target for SSTR5 may help serve to guide the choice of therapeutic treatments.