Thiazolidinediones inhibit REG Ialpha gene transcription in gastrointestinal cancer cells.

REG (Regenerating gene) Ialpha protein functions as a growth factor for gastrointestinal cancer cells, and its mRNA expression is strongly associated with a poor prognosis in gastrointestinal cancer patients. We here demonstrated that PPARgamma-agonist thiazolidinediones (TZDs) inhibited cell proliferation and REG Ialpha protein/mRNA expression in gastrointestinal cancer cells. TZDs inhibited the REG Ialpha gene promoter activity, via its cis-acting element which lacked PPAR response element and could not bind to PPARgamma, in PPARgamma-expressing gastrointestinal cancer cells. The inhibition was reversed by co-treatment with a specific PPARgamma-antagonist GW9662. Although TZDs did not inhibit the REG Ialpha gene promoter activity in PPARgamma-non-expressing cells, PPARgamma overexpression in the cells recovered their inhibitory effect. Taken together, TZDs inhibit REG Ialpha gene transcription through a PPARgamma-dependent pathway. The TZD-induced REG Ialpha mRNA reduction was abolished by cycloheximide, indicating the necessity of novel protein(s) synthesis. TZDs may therefore be a candidate for novel anti-cancer drugs for patients with gastrointestinal cancer expressing both REG Ialpha and PPARgamma.

Recombinant proprotein convertase 4 (PC4) from Leishmania tarentolae expression system: purification, biochemical study and inhibitor design.

Proprotein convertase 4 (PC4) is a member of Ca2+-dependent mammalian subtilases called Proprotein convertases (PCs) or Proprotein convertases subtilisin kexin (PCSK). PC4 plays a key role in mammalian fertilization, sperm maturation and sperm-egg fusion. Full length and C-terminal truncated rPC4 have been expressed using Leishmania tarentolae expression system. Secreted soluble enzyme was recovered in good yield from concentrate medium and purified by DEAE anion exchange and arginine-agarose column chromatographies. This is the first attempt to produce rec (recombinant) PC4 by Leishmania expression system in reasonably pure and enzymatically active form. The eluted fraction contained PC4 protein as confirmed by immunoreactivity using PC4-specific antibodies. Two protein bands at approximately 62, 53 kDa in SDS-PAGE were attributed to C-terminal truncated PC4 forms. The fraction displayed strong protease activity towards fluorogenic Boc-RVRR-MCA and various intramolecularly quenched peptides derived from PC4-substrates. It also cleaved proIGF-2 to produce active IGF-2 confirming its role in this maturation process. Moreover PC4-mediated proteolysis was efficiently blocked by a newly designed prodomain rPC4(101-116) peptide with IC(50) in low microM level. Similar but more potent PC4-inhibitory activity with K(i) in low nM range was observed with the tetrapeptide chloromethyl ketones, Dec-RVKR/K-cmk (chloromethyl ketone). The study showed that such PC4 inhibitors may find potential therapeutic and clinical applications in male fertility.

Cyclin D1 activation through ATF-2 in Reg-induced pancreatic beta-cell regeneration.

Regenerating gene product (Reg) is induced in pancreatic beta-cells and acts as an autocrine/paracrine growth factor for regeneration via a cell surface Reg receptor. However, the manner by which Reg induces beta-cell regeneration was unknown. In the present study, we found that Reg increased phospho-ATF-2, which binds to -57 to -52 of the cyclin D1 gene to activate the promoter. The Reg/ATF-2-induced cyclin D1 promoter activation was attenuated by PI(3)K inhibitors such as LY294002 and wortmannin. In Reg knockout mouse islets, the levels of phospho-ATF-2, cyclin D1, and phospho-Rb were greatly decreased. These results indicate that the Reg-Reg receptor system stimulates the PI(3)K/ATF-2/cyclin D1 signaling pathway to induce beta-cell regeneration.

Molecular cloning, expression and chromosomal localization of a novel human REG family gene, REG III.

Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes [Diabetes 51(Suppl. 3) (2002) S462]. Plural type III Reg genes were found in mouse and rat. On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human. In the present study, we found a novel human type III REG gene, REG III. This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP. REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine. IL-6 responsive elements existed in the 5'-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis. All the human REG family genes identified so far (REG Ialpha, REG Ibeta, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158-175-aa secretory proteins. By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12. The gene order from centromere to telomere was 5' HIP/PAP 3'-5' RS 3'-3' REG Ialpha 5'-5' REG Ibeta 3'-3' REG III 5'. These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region.