Transgenic overexpression of hepatocyte growth factor in the beta-cell markedly improves islet function and islet transplant outcomes in mice.

Recent advances in human islet transplantation have highlighted the need for expanding the pool of beta-cells available for transplantation. We have developed three transgenic models in which growth factors (hepatocyte growth factor [HGF], placental lactogen, or parathyroid hormone-related protein) have been targeted to the beta-cell using rat insulin promoter (RIP). Each displays an increase in islet size and islet number, and each displays insulin-mediated hypoglycemia. Of these three models, the RIP-HGF mouse displays the least impressive phenotype under basal conditions. In this study, we show that this mild basal phenotype is misleading and that RIP-HGF mice have a unique and salutary phenotype. Compared with normal islets, RIP-HGF islets contain more insulin per beta-cell (50 +/- 5 vs. 78 +/- 9 ng/islet equivalent [IE] in normal vs. RIP-HGF islets, P < 0.025), secrete more insulin in response to glucose in vivo (0.66 +/- 0.06 vs. 0.91 +/- 0.10 ng/ml in normal vs. RIP-HGF mice, P < 0.05) and in vitro (at 22.2 mmol/l glucose: 640 +/- 120.1 vs. 1,615 +/- 196.9 pg. microg protein(-1). 30 min(-1) in normal vs. RIP-HGF islets, P < 0.01), have two- to threefold higher GLUT2 and glucokinase steady-state mRNA levels, take up and metabolize glucose more effectively, and most importantly, function at least twice as effectively after transplantation. These findings indicate that HGF has surprisingly positive effects on beta-cell mitogenesis, glucose sensing, beta-cell markers of differentiation, and transplant survival. It appears to have a unique and unanticipated effective profile as an islet mass- and function-enhancing agent in vivo.

Decreased levels of the androgen receptor in the mature rat phallus are associated with decreased levels of androgen receptor messenger ribonucleic acid.

Growth of the penis at sexual maturation is under the control of androgens, but growth ceases as the organ reaches adult size despite continued high levels of circulating androgen. Previous studies have shown that the cessation of penile growth is associated with a decrease in the quantity of androgen receptor, as detected by ligand binding and immunological methods. In the current studies we demonstrate that the decreased androgen receptor levels correlate with a decrease in androgen receptor mRNA content in the penile corpus and os, but not in the glans penis. These findings suggest that modulation of androgen receptor mRNA levels in the body of the penis may be important to the control of androgen-dependent growth in the tissue, and that the control of androgen receptor mRNA levels differs among the different cell types that comprise the penis. To explore the mechanisms controlling androgen receptor expression, we examined the transcription initiation site of the rat androgen receptor gene in ventral prostate and in three compartments of the penis: the corpus, the urethra, and the glans penis. The same promoter is employed in all preparations, suggesting that the different patterns of androgen receptor mRNA expression in these tissues with age are controlled by factors that modulate the activity of the same promoter.

Androgen receptor levels in the rat penis are controlled differently in distinctive cell types.

The rat phallus grows during sexual maturation as serum androgen concentrations rise to adult levels, and growth ceases when sexual maturity is attained despite the continued presence of adult serum androgen levels. This cessation of growth is correlated temporally with a diminution in the levels of androgen receptor, as detected by assays of ligand binding. To determine whether the change in androgen receptor content occurs in all cells in the tissue, immunohistochemical studies of the penile androgen receptor were performed in rats of different ages. In immature animals and animals castrated prepubertally, the androgen receptor is detected in virtually all cell types, including the corpus cavernosum penis, the small lateral cavernous bodies, the corpus cavernosum urethra, skin, urethra, and os penis. By contrast, in the mature rat penis minimal androgen receptor is evident within the corporal tissue and os, although immunoreactivity remains detectable in the penile skin and urethra. It is concluded that the cessation of androgen-mediated growth correlates with a decrease in androgen receptor levels in the body of the penis.

The C-terminal region of PTHrP, in addition to the nuclear localization signal, is essential for the intracrine stimulation of proliferation in vascular smooth muscle cells.

PTHrP is secreted by most cell types. In addition to a paracrine/autocrine role, PTHrP has "intracrine" actions, entering the nuclear compartment under the direction of a classic bipartite nuclear localization signal. In vascular smooth muscle cells, nuclear entry stimulates mitogenesis. In the current study, we sought to more precisely define the regions of PTHrP required for the activation of mitogenesis in vascular smooth muscle cells. PTHrP deletion mutants missing large regions [i.e. the signal peptide, N terminus (1--36), mid region (38--86), nuclear localization signal, C terminus (108--139), or combinations of the above] were expressed in A-10 vascular smooth muscle cells. The consequences on nuclear localization and proliferation were examined. Deletion of the nuclear localization signal prevented nuclear entry and slowed proliferation. Deletion of the highly conserved N terminus or mid region had no impact on nuclear localization or on proliferation. Deletion of the C terminus had no deleterious effect on nuclear localization but dramatically reduced proliferation. Thus, the nuclear localization signal is both necessary and sufficient for nuclear localization of PTHrP. In contrast, activation of proliferation in vascular smooth muscle cells requires both an intact nuclear localization signal and an intact C terminus. Whereas the nuclear localization signal is required for nuclear entry, the C terminus may serve a trans-activating function to stimulate mitogenesis once inside the nucleus of vascular smooth muscle cells.

Using beta-cell growth factors to enhance human pancreatic Islet transplantation.

This is a particularly exciting time in the field of pancreatic islet growth, development, and survival. The recent publication of a study demonstrating that human pancreatic islet transplantation is both technically and immunologically feasible has highlighted the need for large supplies of pancreatic islets or pancreatic beta cells for larger-scale islet transplantation in patients with diabetes. This, together with a rapid expansion in the past several years of the understanding of mechanisms of islet growth, development, and survival, has accelerated and invigorated efforts to therapeutically harness the cellular mechanisms responsible for pancreatic beta-cell proliferation, survival, and development and to take advantage of this new knowledge to enhance the availability, survival, and function of pancreatic beta cells in human islet transplantation for diabetes mellitus. Here, we briefly review the confluence of events that have provided optimism and energy to the islet transplant field, and we focus on peptide growth factors that eventually may be deployed in the effort to augment islet mass and function in patients with diabetes.

Functional analysis of the human androgen receptor promoter.

Although androgen receptor levels vary widely during development, our previous studies have suggested that a single promoter is active in a number of human and rat tissues and cell lines. To examine the elements controlling androgen receptor expression, we performed deletion mapping and site-directed mutagenesis of the human androgen receptor promoter and assayed these promoter fusions in human cell lines that produce the androgen receptor as well as those deficient in the androgen receptor, both in the presence and absence of androgen. Our studies identify a region (-74 to +87) surrounding the site of transcription initiation that constitutes the core of the androgen receptor promoter. When assayed in T47D cells (an AR-expressing cell line), this segment was sufficient to direct the expression of reporter genes at levels similar to that observed for larger promoter fragments, and further deletions led to an appreciable loss of promoter activity. DNA sequences surrounding this region appear to modulate the activity of this minimal promoter in a cooperative fashion. Site-directed mutagenesis and mobility shift assays demonstrated the importance of regulatory regions upstream of the transcription initiation site including an SP1 binding site and two segments with similarities to consensus HLH protein binding sites (E-boxes). Androgen treatment did not cause a decrease in activity of any of the transiently transfected promoter fusions tested, suggesting that the promoter does not contain the information necessary for autoregulation or that a posttranscriptional or posttranslational mechanism is responsible for the regulation of AR mRNA by ligand. Furthermore, the androgen receptor promoter fusions displayed differing transcriptional activities when transfected into two cell lines deficient in androgen receptor expression suggesting heterogeneity in the mechanisms responsible for this deficiency.

Androgen receptor of rat penis is down-regulated by androgen.

To provide insight into the factors that control androgen receptor levels in rat penis, we assessed 5 alpha-[3H]-dihydrotestosterone binding in low-salt [10 mM tris(hydroxymethyl)aminomethane (Tris), 10 mM Na2M0O4] and high-salt (10 mM Tris, 10 mM Na2M0O4, 0.5 M KCl) extracts of rat penis using sucrose density gradients. Total receptor content decreased from approximately 729 +/- 114 fmol/g tissue at 3 wk of age to less than 50 fmol/g tissue at 10 wk of age. Castration of 3-wk-old rats prevented penile growth and the age-related decline in penile androgen receptor. Treatment of 3-wk-old castrated rats with 5 alpha-dihydrotestosterone caused an acceleration in the decline in receptor levels compared with intact animals. Castration of 10-wk-old rats (after androgen receptor levels had decreased) did not result in an increase in the amount of total androgen receptor by 16 wk of age. To determine the specificity of the androgen-mediated decline in receptor levels, the amounts of prostate androgen receptor were compared with those of the penis at different ages. When expressed as femtomoles per organ, the total androgen receptor level in the prostate increased fourfold from 3 to 10 wk of age, whereas the total androgen receptor in the penis declined approximately threefold. We conclude that the downregulation of the penile androgen receptor content that occurs in the rat between 3 and 10 wk of age is androgen mediated, does not occur in all androgen target tissues, and is prevented but not reversed by castration.

Hepatocyte growth factor overexpression in the islet of transgenic mice increases beta cell proliferation, enhances islet mass, and induces mild hypoglycemia.

Hepatocyte growth factor (HGF) is produced in pancreatic mesenchyme-derived cells and in islet cells. In vitro, HGF increases the insulin content and proliferation of islets. To study the role of HGF in the islet in vivo, we have developed three lines of transgenic mice overexpressing mHGF using the rat insulin II promoter (RIP). Each RIP-HGF transgenic line displays clear expression of HGF mRNA and protein in the islet. RIP-mHGF mice are relatively hypoglycemic in post-prandial and fasting states compared with their normal littermates. They display inappropriate insulin production, striking overexpression of insulin mRNA in the islet, and a 2-fold increase in the insulin content in islet extracts. Importantly, beta cell replication rates in vivo are two to three times higher in RIP-HGF mice. This increase in proliferation results in a 2-3-fold increase in islet mass. Moreover, the islet number per pancreatic area was also increased by approximately 50%. Finally, RIP-mHGF mice show a dramatically attenuated response to the diabetogenic effects of streptozotocin. We conclude that the overexpression of HGF in the islet increases beta cell proliferation, islet number, beta cell mass, and total insulin production in vivo. These combined effects result in mild hypoglycemia and resistance to the diabetogenic effects of streptozotocin.