Expression of reg/PSP, a pancreatic exocrine gene: relationship to changes in islet beta-cell mass

A cDNA termed reg was recently isolated by differential screening of a library prepared from regenerating islets isolated from pancreatic remnants of rats subjected to 90% pancreatectomy and nicotinamide treatment. This led to speculation that this gene may be involved in expansion of beta-cell mass. In the current study we have measured reg expression after implantation and resection of a solid insulinoma tumor into rats, maneuvers known, respectively, to reduce and reexpand the volume of beta-cells in the islet. Animals with an implanted insulinoma tumor became profoundly hypoglycemic. Islet beta-cells declined from the normal 75% of total islet volume to less than 30%, in concert with a marked reduction in the reg mRNA level. Removal of the tumor resulted in a sharp increase in beta-cell replication, as measured by [3H]thymidine incorporation and a return to normal beta-cell volume within 4 days of tumor resection. This was associated with a transient induction in reg expression compared to that in tumor-bearing animals, effectively returning the amount of reg mRNA to the levels found in normal animals within 48 h; at later time points after tumor removal (3-7 days) reg expression declined, but then rose toward normal. In situ hybridization analysis localized the initial induction in reg mRNA expression to the exocrine pancreas. Continuous infusion of insulin into normal rats for 4 days, a maneuver that does not significantly reduce beta-cell mass, resulted in dramatically reduced insulin mRNA in islets, but no change in the levels of reg mRNA. We conclude that the diminution in pancreatic beta-cell mass caused by subcutaneous implantation of an insulinoma is associated with reduced reg gene expression and that the increase in beta-cell replication after resection of the tumor is preceded by return of reg gene expression toward normal.     

Growth hormone is a growth factor for the differentiated pancreatic beta-cell

The regulation of the growth of the pancreatic beta-cell is poorly understood. There are previous indications of a role of GH in the growth and insulin production of the pancreatic islets. In the present study we present evidence for a direct long-term effect of GH on proliferation and insulin biosynthesis of pancreatic beta-cells in monolayer culture. In culture medium RPMI 1640 supplemented with 2% normal human serum islets or dissociated islet cells from newborn rats maintained their insulin-producing capacity. When supplemented with 1-1000 ng/ml pituitary or recombinant human GH the islet cells attached, spread out, and proliferated into monolayers mainly consisting of insulin-containing cells. The number of beta-cells in S-phase was increased from 0.9-6.5% as determined by immunochemical staining of bromodeoxyuridine incorporated into insulin-positive cells. The increase in cell number was accompanied with a continuous increase in insulin release to the culture medium reaching a 10- 20-fold increase after 2-3 months with a half-maximal effect at about 10 ng/ml human GH. The biosynthesis of (pro)insulin was markedly increased with a normal rate of conversion of proinsulin to insulin. It is concluded that GH is a potent growth factor for the differentiated pancreatic beta-cell.     

Identification of the non-specific cytotoxic cell receptor protein 1 (NCCRP1) in regenerating axolotl limbs

The teleost non-specific cytotoxic cells (NCC) are evolutionary precursors of the mammalian natural killer (NK) cells and an important element of innate immunity. The non-specific cytotoxic cell receptor protein (NCCRP1) is a characteristic cell surface protein with main functions in target cell recognition and cytotoxicity with sequence information available for many species of fish. We have isolated a cDNA encoding the Axolotl homologue of fish NCCRP1 out of limb regeneration blastema and analysed its expression by RT-PCR. Sequence analysis revealed a high degree of homology with teleost NCCRP1 on nucleotide and deduced amino acid levels. NCCRP1 contains a conserved C-terminal F-box-associated domain (FBA) and proline-rich motifs (PRM) characteristic for this protein family. NCCRP1 is expressed in multiple tissues with high levels in limb regeneration blastema. The present work describes for the first time the cloning of the NCCRP1 gene in a tetrapod vertebrate providing a valuable link between fish and higher vertebrates. Our findings suggest the existence of NCC in axolotl and a role of the innate immune system in the processes of limb regeneration.     

A role for G(z) in pancreatic islet beta-cell biology

Glucose-stimulated insulin secretion and beta-cell growth are important facets of pancreatic islet beta-cell biology. As a result, factors that modulate these processes are of great interest for the potential treatment of Type 2 diabetes. Here, we present evidence that the heterotrimeric G protein G(z) and its effectors, including some previously thought to be confined in expression to neuronal cells, are present in pancreatic beta-cells, the largest cellular constituent of the islets of Langerhans. Furthermore, signaling pathways upon which G alpha(z) impacts are intact in beta-cells, and G alpha(z) activation inhibits both cAMP production and glucose-stimulated insulin secretion in the Ins-1(832/13) beta-cell-derived line. Inhibition of glucose-stimulated insulin secretion by prostaglandin E (PGE1) is pertussis-toxin insensitive, indicating that other G alpha(i) family members are not involved in this process in this beta-cell line. Indeed, overexpression of a selective deactivator of G alpha(z), the RGS domain of RGSZ1, blocks the inhibitory effect of PGE1 on glucose-stimulated insulin secretion. Finally, the inhibition of glucose-stimulated insulin secretion by PGE1 is substantially blunted by small interfering RNA-mediated knockdown of G alpha(z) expression. Taken together, these data strongly imply that the endogenous E prostanoid receptor in the Ins-1(832/13) beta-cell line couples to G(z) predominantly and perhaps even exclusively. These data provide the first evidence for G(z) signaling in pancreatic beta-cells, and identify an endogenous receptor-mediated signaling process in beta-cells that is dependent on G alpha(z) function.     

Expression and localization of regenerating gene I in a rat liver regeneration model

Regenerating gene (Reg) I has been identified as a regenerative/proliferative factor for pancreatic islet cells. We examined Reg I expression in the regenerating liver of a rat model that had been administered 2-acetylaminofluorene and treated with 70% partial hepatectomy (2-AAF/PH model), where hepatocyte and cholangiocyte proliferation was suppressed and the hepatic stem cells and/or hepatic progenitor cells were activated. In a detailed time course study of activation of hepatic stem cells in the 2-AAF/PH model, utilizing immunofluorescence staining with antibodies of Reg I and other cell-type-specific markers, we found that Reg I-expressing cells are present in the bile ductules and increased during regeneration. Reg I-expressing cells were colocalized with CK19, OV6, and AFP. These results demonstrate that Reg I is significantly upregulated in the liver of the 2-AAF/PH rat model, accompanied by the formation of bile ductules during liver regeneration.     

Characterization of a purified chromatin acceptor protein (receptor binding factor 1) for the avian oviduct progesterone receptor

The specific, high-affinity binding of the avian oviduct progesterone receptor (PR) with target-cell nuclei and chromatin has been shown to involve DNA complexed with specific chromatin acceptor proteins. One of these chromatin acceptor proteins has been partially purified and found to be a small hydrophobic protein with a broad pI of 5.0-6.0 [Goldberger, A., & Spelsberg, T. C., (1988) Biochemistry 27, 2103-2109]. This paper describes the final purification over 100,000-fold to apparent homogeneity of this candidate PR acceptor protein, termed the receptor binding factor 1 (RBF-1). When the avian genomic DNA is bound by RBF-1, saturable, high-affinity (KD approximately 2 x 10(-9) M) binding sites for PR are generated. RBF-1 has a unique, hydrophobic N-terminal sequence. The PR binding to the RBF-1-DNA complexes is shown to be dependent on an intact activated PR with which excess nonradiolabeled PR can compete. By use of a new, highly specific monoclonal antibody (mAb) to the RBF-1 with Western immunoblotting, RBF-1 was shown to be localized in the nucleus and to be tissue and species specific. Selective removal of the chromatin proteins containing RBF-1 results in the loss of the highest affinity class of PR binding sites. A second class of residual PR binding sites remains in the nucleoacidic protein (NAP), a complex of proteins more tightly bound to the DNA. This class of PR binding activity has been classified as the RBF-2. The RBF-1 is estimated to be 0.03% of the total chromatin protein with about 1.2 x 10(5) molecules/diploid cell.     

RIP-Cre revisited, evidence for impairments of pancreatic beta-cell function

The Cre/loxP recombinase system for performing conditional gene targeting experiments has been very useful in exploring genetic pathways that control both the development and function of pancreatic beta-cells. One particular line of transgenic mice (B6.Cg-Tg(Ins2-cre)25Mgn/J), commonly called RIP-Cre, in which expression of Cre recombinase is controlled by a short fragment of the rat insulin II gene promoter has been used in at least 21 studies on at least 17 genes. In most of these studies inactivation of the gene of interest was associated with either glucose intolerance or frank diabetes. Experimental evidence has been gradually emerging to suggest that RIP-Cre mice alone display glucose intolerance. In this study experiments from three laboratories demonstrate that RIP-Cre mice, in the absence of genes targeted by loxP sites, are glucose intolerant, possibly due to impaired insulin secretion. In addition, we review the use of RIP-Cre mice and discuss possible molecular underpinnings and ramifications of our findings.     

Identification of a novel gene (ADPRTL1) encoding a potential Poly(ADP-ribosyl)transferase protein

Poly(ADP-ribosyl)ation of nuclear proteins plays a significant role in the maintenance of genomic DNA stability. To date, four poly(ADP-ribosyl)ating proteins have been identified in humans. We now report the full-length sequence, expression profile, and chromosomal localization of a novel gene, ADPRTL1, encoding an ADP-ribosyltransferase-like protein. The predicted open reading frame encodes a protein of 1724 amino acids with a molecular mass of 192.8 kDa. The protein contains a region showing homology to the catalytic domains of the nuclear-localized ADP-ribosyltransferase proteins (Adprt), two recently identified Adprt-like proteins (Adprtl2 and Adprtl3), and the telomere-associated protein tankyrase. Key amino acids known to be important for the activity of these enzymes are conserved within this region of the Adprtl1 protein, indicating that Adprtl1 is a functional poly(ADP-ribosyl)transferase. As has been noted for tankyrase, sequence analysis of the Adprtl1 protein suggests that it is not capable of binding DNA directly. Thus, the transferase activity of Adprtl1 may be activated by other factors such as protein-protein interaction mediated by the extensive carboxyl terminus. We have subsequently refined the location of the ADPRTL1 genomic locus to 13q11, close to the recently cloned ZNF198 gene.     

Multiple gene action determining a mouse salivary protein phenotype: Identification of the structural gene for androgen binding protein (Abp)

A novel variation in electrophoretic phenotype is described for a mouse salivary androgen binding protein (Abp). Crosses show that the variation is inherited in an autosomal codominant manner and protein characterization studies show that the variant Abp differs in isoelectric point from the common form of the protein. Those observations suggest that the variation involves the structural gene for the mouse salivary Abp. The genetic studies also show that the electrophoretic mobility of the variant Abp can be influenced by the sex-limited saliva pattern (Ssp) gene. The Ssp ^S allele alters the electrophoretic mobility of Abp in males at puberty or in females which have received exogenous testosterone [Karn, R. C., Dlouhy, S. R., Springer, K. R., Hjorth, J. P., and Nielsen, J. T. (1982). Biochem Genet. 20:493]. This study shows that Abp and Ssp are distinct genes which are not closely linked and that Ssp ^S is trans active in F₁ (Abp ^a /Abp ^b , Ssp ^S /Ssp ^F ) males.SRD was supported in part by PHS General Medical Training Grant T32 GMO7468 and the Indiana University School of Medicine Research Program in Academic Medicine. RCK was supported in part by PHS Career Development Award 1 KO4 AMOO284.     

Identification of a new Bradyrhizobium japonicum gene (frxA) encoding a ferredoxinlike protein.

An open reading frame of 74 codons was identified downstream of the nifB gene of Bradyrhizobium japonicum 110. The predicted amino acid sequence shared 63% similarity with the Rhodopseudomonas palustris ferredoxin I sequence. We propose to name the gene frxA. The frxA gene was found to be cotranscribed with the nifB gene. An insertion mutation within frxA hardly affected nitrogen fixation activity.     

Identification of the human Mnk2 gene (MKNK2) through protein interaction with estrogen receptor beta

We have identified and characterized the human Mnk2 gene (HGMW-approved gene symbol MKNK2) through a yeast two-hybrid screen in which the Mnk2 protein interacted with the ligand-binding domain of estrogen receptor beta (ERbeta). Human Mnk2 is homologous to murine Mnk2 ( approximately 94% identical) and human Mnk1 (71% identical), both of which encode MAP kinase interacting kinases that are phosphorylated and activated by ERK1 and 2. This report presents a thorough genomic sequence analysis revealing that the human Mnk2 gene has two C-terminal splice variants, designated here as Mnk2a and Mnk2b. These two isoforms are identical over the first 385 amino acids of the coding sequence and differ only in the final exon which encodes an additional 80 residues for Mnk2a and 29 residues for Mnk2b. A more detailed biological analysis in yeast showed that the Mnk2 interaction was selective for ERbeta as opposed to ERalpha and that the interaction was specific to Mnk2b as opposed to Mnk2a or Mnk1. This pattern was reproduced in a mammalian two-hybrid system using a completely different set of fusion partners; and in both yeast and mammalian systems, the addition of estradiol decreased the interaction. While it remains unknown whether ERbeta is a substrate of Mnk2, the interaction of these two proteins is reminiscent of ERalpha and ribosomal S6 kinase (p90-RSK), another MAP kinase-regulated kinase homologous to Mnk2 that is known to phosphorylate ERalpha.     

Nephroblastoma overexpressed gene (NOV) codes for a growth factor that induces protein tyrosine phosphorylation

NOV (nephroblastoma overexpressed gene) is a member of the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, NOV) family of proteins. These proteins are cysteine-rich and are noted for having growth-regulatory functions. We have isolated the rat NOV gene, and the DNA sequence shares 90% identity with the mouse and 80% identity with the human sequences. The rat NOV gene was expressed in all rat tissues examined, including brain, lung, heart, kidney, liver, spleen, thymus and skeletal muscle. Higher levels of rat NOV mRNA were seen in the brain, lung and skeletal muscle compared to the other tissues. Examination of NOV expression in various human cell lines revealed that NOV was expressed in U87, 293, T98G, SK-N-MC and Hs683 but not in HepG2, HL60, THP1 and Jurkat. The human NOV gene was transfected into 293 cells and the expressed protein purified. When 3T3 fibroblasts were treated with this recombinant NOV protein, a dose-dependent increase in proliferation was observed. Analysis of tyrosine-phosphorylated proteins revealed that when 3T3 cells were treated with NOV, a 221 kDa protein was phosphorylated. These data suggest that NOV can act as a growth factor for some cells and binds to a specific receptor that leads to the phosphorylation of a 221 kDa protein.