Arunangsu Dey

Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects

The subtilisin-like proprotein convertases PC1/3 (SPC3) and PC2 (SPC2) are believed to be the major endoproteolytic processing enzymes of the regulated secretory pathway. They are expressed together or separately in neuroendocrine cells throughout the brain and dispersed endocrine system in both vertebrates and invertebrates. Disruption of the gene-encoding mouse PC1/3 has now been accomplished and results in a syndrome of severe postnatal growth impairment and multiple defects in processing many hormone precursors, including hypothalamic growth hormone-releasing hormone (GHRH), pituitary proopiomelanocortin to adrenocorticotropic hormone, islet proinsulin to insulin and intestinal proglucagon to glucagon-like peptide-1 and -2. Mice lacking PC1/3 are normal at birth, but fail to grow normally and are about 60% of normal size at 10 weeks. They lack mature GHRH, have low pituitary growth hormone (GH) and hepatic insulin-like growth factor-1 mRNA levels and resemble phenotypically the “little” mouse (Gaylinn, B. D., Dealmeida, V. I., Lyons, C. E., Jr., Wu, K. C., Mayo, K. E. & Thorner, M. O. (1999) Endocrinology 140, 5066–5074) that has a mutant GHRH receptor. Despite a severe defect in pituitary proopiomelanocortin processing to mature adrenocorticotropic hormone, blood corticosterone levels are essentially normal. There is marked hyperproinsulinemia but without impairment of glucose tolerance. In contrast, PC2-null mice lack mature glucagon and are chronically hypoglycemic (Furuta, M., Yano, H., Zhou, A., Rouille, Y., Holst, J., Carroll, R., Ravazzola, M., Orci, L., Furuta, H. & Steiner, D. (1997) Proc. Natl. Acad. Sci. USA 94, 6646–6651). The PC1/3-null mice differ from a human subject reported with compound heterozygosity for defects in this gene, who was of normal stature but markedly obese from early life. The PC1/3-null mice are not obese. The basis for these phenotypic differences is an interesting topic for further study. These findings prove the importance of PC1/3 as a key neuroendocrine convertase.

No mantle formation in rodent islets—The prototype of islet revisited

Emerging reports on human islets emphasize distinct differences from the widely accepted prototype of rodent islets, raising questions over their suitability for human studies. Here we aim at elucidating architectural differences and similarities of human versus rodent islets. The cellular composition and architecture of human and rodent islets were compared through three-dimensional (3D) reconstructions. Physiological and pathological changes were examined using islets from various mouse models such as non-obese diabetic (NOD), ob/ob, db/db mice and during pregnancy. A subpopulation of human islets is composed of clusters of alpha-cells within the central beta-cell cores, while the overall proportion of alpha-cells varies among islets. In mouse islets under normal conditions, alpha-cells are localized in the islet periphery, but they do not envelop the entire beta-cell core, so that beta-cells are exposed on the outer layer of the islet, as in most human islets. Also, an increased proportion of alpha-cells within the central core is observed in the pancreas of mouse models exhibiting increased demand for insulin. In summary, human and mouse islets share common architectural features as endocrine micro-organs. Since these may hold a key to better understanding islet plasticity, our concept of the prototypic islet should be revised.

Processing of cocaine- and amphetamine-regulated transcript (CART) precursor proteins by prohormone convertases (PCs) and its implications.

Cocaine- and amphetamine-regulated transcript (CART) peptides are expressed in several neuroendocrine tissues, including hypothalamus, pituitary, gut, adrenal and pancreas, and are involved in regulating important biological processes including feeding/appetite, drug reward and stress. CART is synthesized as larger, inactive peptide precursors (pro-CART) that require endoproteolytic processing to generate smaller, active forms. Prohormone/proprotein convertases (PCs), a family of calcium-dependent, serine endoproteases, have been shown to cleave many protein precursors in the regulated/constitutive secretory pathway to generate smaller fragments. In our previous studies, we have demonstrated the important roles of the two neuroendocrine-specific PCs, PC2 and PC1/3, in processing the two pro-CART isoforms, long (102aa) and short (89aa), to generate the bioactive CART peptides, I (55-102/42-89) and II (62-102/49-89) as well as the intermediate fragments, 10-89 and 33-102. Our subsequent studies have revealed the participation of another PC family member, PC5/6A (the soluble isoform of a widely expressed PC, PC5/6), in cleaving both precursor isoforms. We conclude that PC5/6A contributes to the normal efficient processing of pro-CART and is functionally more redundant with PC2 than PC1/3 in generating both CART I and II.

GSK-3 inactivation or depletion promotes β-cell replication via down regulation of the CDK inhibitor, p27 (Kip1)

Diabetes (T1DM and T2DM) is characterized by a deficit in β-cell mass. A broader understanding of human β-cell replication mechanism is thus important to increase β-cell proliferation for future therapeutic interventions. Here, we show that p27 (Kip1), a CDK inhibitor, is expressed abundantly in isolated adult human islets and interacts with various positive cell cycle regulatory proteins including D-type cyclins (D1, D2 and D3) and their kinase partners, CDK4 and CDK6. Also, we see interaction of cyclin E and its kinase partner, CDK2, with p27 suggesting a critical role of p27 as a negative cell cycle regulator in human islets. Our data demonstrate interaction of p27 with GSK-3 in β-cells and show, employing rodent β-cells (INS-1), isolated human islets and purified β-cells derived from human islets, that siRNA-mediated depletion of GSK-3 or p27 or 1-AKP / BIO – mediated GSK-3 inhibition results in increased β-cell proliferation. We also see reduction of p27 levels following GSK-3 inactivation or depletion. Our data show that serum induction of quiescent INS-1 cells leads to sequential phosphorylation of p27 on its S10 and T187 residues with faster kinetics for S10 corresponding with the decreased levels of p27. Altogether our findings indicate that p27 levels in β-cells are stabilized by GSK-3 and thus p27 down regulation following GSK-3 depletion / inactivation plays a critical role in promoting β-cell replication.

Gamma Interferon Induces Expression of Mad1 Gene in Macrophage, Which Inhibits Colony-Stimulating Factor-1-Dependent Mitogenesis

Gamma interferon (IFNγ) has long been known as an antiproliferative cytokine. The mechanism of its action, however, remains elusive. Monocytes and macrophages are primary targets of IFNγ. To understand the antiproliferative signaling of IFNγ, we studied the effect of IFNγ on expression of c‐Myc, Mad1, Max, cyclin D1, and cyclin D2 genes in both a macrophage cell line and in primary bone marrow–derived macrophages (BMM) in response to colony‐stimulating factor‐1 (CSF‐1). We found that whereas IFNγ inhibits CSF‐1–stimulated c‐Myc gene expression, it induces Mad1 expression. Induction of Mad1 mRNA could be detected as early as 90 min following IFNγ treatment and was maintained for at least 15 h. These results suggest that IFNγ treatment could shift the Myc–Max complex to the Mad1–Max complex in cells. The levels of Max, cyclin D1, and cyclin D2, however, remained unchanged. Enforced ectopic expression of Mad1 in the cells results in inhibition of [3H]thymidine incorporation and proliferation in response to CSF‐1. This study suggests a mechanism by which IFNγ inhibits CSF‐1–stimulated proliferation of macrophages, i.e., by elevating the Mad1 level in the cells. J. Cell. Biochem. 72:232–241, 1999.

RNAi-mediated silencing of prohormone convertase (PC) 5/6 expression leads to impairment in processing of cocaine- and amphetamine-regulated transcript (CART) precursor.

Understanding the functions of the widely expressed PCs (prohormone/proprotein convertases), including PC5/6, furin and PACE4 (paired basic amino acid cleaving enzyme 4), in animal models is difficult since individual knockouts of these PCs in mice exhibit early embryonic lethality. To investigate the roles of PC5/6 in processing pro-CART (pro-cocaine- and amphetamine-regulated transcript), an important anorexigenic peptide precursor, we have generated GH3 cells silenced for PC5/6 expression by RNAi (RNA interference). We show, following transient knockdown of PC5/6 in these neuroendocrine cells, that generation of the two bioactive forms, CART I (amino acids 42-89/55-102) and CART II (amino acids 49-89/62-102), from pro-CART is impaired due to a lack particularly of the A isoform of PC5/6. The results indicate that PC5/6A shares specificities primarily with PC2 (PC5/6A<PC2) in cleaving the pairs of basic residues, KR (40, 41 //53, 54/) and KK (47, 48//60, 61/), within the pro-CART isoforms [see Dey, Zhu, Carroll, Turck, Stein and Steiner (2003) J. Biol. Chem. 278, 15007-15014]. We do not find any significant role of PC5/6A in processing the RXXR (29-32/) site for production of intermediate CART (amino acids 33-102) from long pro-CART. The findings taken altogether indicate that PC5/6 participates in normal processing of pro-CART.

Cell cycle-independent induction of D1 and D2 cyclin expression, but not cyclin–Cdk complex formation or Rb phosphorylation, by IFNγ in macrophages

During G1 to S phase transition, D-type cyclins form complexes with cyclin-dependent kinases (Cdk), which in turn phosphorylate retinoblastoma gene product (Rb) and inhibit its growth-inhibitory function, leading ultimately to cell proliferation. We report here a novel finding that D1 and D2 cyclins are induced in macrophages by antiproliferative factor gamma interferon (IFNgamma). The induction appears to be transcriptional activation of the D cyclin genes, since indirect events such as IFNgamma-induced colony-stimulating factor-1 (CSF-1) autocrine stimulation, alteration of D1 and D2 mRNA stability and lipopolysaccharide contamination in commercial IFNgamma preparations play no roles. In contrast to CSF-1, IFNgamma neither induces D1-Cdk4 complex formation and Rb hyperphosphorylation nor interferes with CSF-1-stimulated D1-Cdk4 interaction and Rb phosphorylation, while it completely blocks CSF-1-stimulated cell proliferation. This study suggests that induction of D1 and D2 cyclins is not necessarily associated with cell cycle progression, and D cyclins may have cell cycle-independent functions in response to IFNgamma.