Mapping and targeted viral activation of pancreatic nerves in mice reveal their roles in the regulation of glucose metabolism.

A lack of comprehensive mapping of ganglionic inputs into the pancreas and of technology for the modulation of the activity of specific pancreatic nerves has hindered the study of how they regulate metabolic processes. Here we show that the pancreas-innervating neurons in sympathetic, parasympathetic and sensory ganglia can be mapped in detail by using tissue clearing and retrograde tracing (the tracing of neural connections from the synapse to the cell body), and that genetic payloads can be delivered via intrapancreatic injection to target sites in efferent pancreatic nerves in live mice through optimized adeno-associated viruses and neural-tissue-specific promoters. We also show that, in male mice, the targeted activation of parasympathetic cholinergic intrapancreatic ganglia and neurons doubled plasma-insulin levels and improved glucose tolerance, and that tolerance was impaired by stimulating pancreas-projecting sympathetic neurons. The ability to map the peripheral ganglia innervating the pancreas and to deliver transgenes to specific pancreas-projecting neurons will facilitate the examination of ganglionic inputs and the study of the roles of pancreatic efferent innervation in glucose metabolism.

Two-minute versus 6-minute walk distances during 6-minute walk test in neuromuscular disease: Is the 2-minute walk test an effective alternative to a 6-minute walk test?

Functional tests such as Motor Function Measure-32 (MFM-32), supine to stand, ascend/descend stairs permit the assessment of task-specific motor function in neuromuscular disease (NMD). The 6-min walk test (6MWT), though functional, is primarily used to assess endurance and disease progression in children with neuromuscular disorders. Barriers to 6MWT administration, in this population, can include reduced attention span due to age and inability to tolerate test length due to weakness. We propose task-specific functional deficits are related to endurance. Additionally, the 2-min walk test (2MWT) could effectively replace the 6MWT in this population. Seventy-seven participants, ages 5-18, with a variety of neuromuscular disorders performed the 6MWT, timed functional tests (TFT), and the MFM-32. Correlation and paired t-test analyses were used to compare the distance walked in the first 2 min (2MWD) to the distance walked in the entire 6 min (6MWD) and to the functional outcome measures above. The 2MWD strongly correlated with 6MWD and the other outcome measures. Paired t-test analysis also showed that the 2MWD did not differ from the distance walked in the last 2 min of the 6MWT. Although equivalence testing could not reject the claim that this difference exceeded the upper practical limit of 9.5 m, it only showed a modest overestimation of the 4-6MWD compared with the 2MWD. Together, our results support the ability of the 2MWD to predict the 6MWD, specifically in the pediatric neuromuscular disease population.

Lactogens protect rodent and human beta cells against glucolipotoxicity-induced cell death through Janus kinase-2 (JAK2)/signal transducer and activator of transcription-5 (STAT5) signalling.

AIMS/HYPOTHESIS: A leading cause of type 2 diabetes is a reduction in functional beta cell mass partly due to increased beta cell death, triggered by stressors such as glucolipotoxicity (GLT). This study evaluates the hypothesis that lactogens can protect beta cells against GLT and examines the mechanism behind the pro-survival effect. METHODS: The effect of exogenous treatment or endogenous expression of lactogens on GLT-induced beta cell death was examined in INS-1 cells, and in rodent and human islets. The mechanism behind the pro-survival effect of lactogens was determined using an inhibitor, siRNAs, a dominant negative (DN) mutant, and Cre-lox-mediated gene deletion analysis. RESULTS: Lactogens significantly protect INS-1 and primary rodent beta cells against GLT-induced cell death. The pro-survival effect of lactogens in rodent beta cells is mediated through activation of the Janus kinase-2 (JAK2)/signal transducer and activator of transcription-5 (STAT5) signalling pathway. Lactogen-induced increase in the anti-apoptotic B cell lymphoma-extra large (BCLXL) protein is required to mediate its pro-survival effects in both INS-1 cells and primary rodent beta cells. Most importantly, lactogens significantly protect human beta cells against GLT-induced cell death, and their pro-survival effect is also mediated through the JAK2/STAT5 pathway. CONCLUSIONS/INTERPRETATION: These studies, together with previous work, clearly demonstrate the pro-survival nature of lactogens and identify the JAK2/STAT5 pathway as an important mediator of this effect in both rodent and human beta cells. Future studies will determine the effectiveness of this peptide in vivo in the pathophysiology of type 2 diabetes.

Systemic and acute administration of parathyroid hormone-related peptide(1-36) stimulates endogenous beta cell proliferation while preserving function in adult mice.

AIMS/HYPOTHESIS: A major focus in the treatment of diabetes is to identify factors that stimulate endogenous beta cell growth while preserving function. The first 36 amino acids of parathyroid hormone-related protein (PTHrP) are sufficient to enhance proliferation and function in rodent and human beta cells in vitro. This study examined whether acute and systemic administration of the amino-terminal PTHrP(1-36) peptide can achieve similar effects in rodent beta cells in vivo. METHODS: Adult male mice were injected with 40, 80 or 160 µg of PTHrP(1-36) per kg body weight or with vehicle for 25 days. Glucose and beta cell homeostasis, as well as expression of differentiation markers and cell cycle genes were analysed. RESULTS: All three doses of PTHrP(1-36) significantly enhanced beta cell proliferation in vivo at day 25, with 160 µg/kg PTHrP(1-36) increasing proliferation as early as day 5. Importantly, the two higher doses of PTHrP(1-36) caused a significant 30% expansion of beta cell mass, with a short-term improvement in glucose tolerance. PTHrP(1-36) did not cause hypercalcaemia, or change islet number, beta cell size, beta cell death or expression of differentiation markers. Analysis of islet G1/S cell cycle proteins revealed that chronic overabundance of PTHrP(1-139) in the beta cell significantly increased the cell cycle activator cyclin D2 and decreased levels of cyclin-dependent kinase 4 inhibitor (p16( Ink4a ) [Ink4a also known as Cdkn2a]), but acute treatment with PTHrP(1-36) did not. CONCLUSIONS/INTERPRETATION: Acute and systemic administration of PTHrP(1-36) increases rodent beta cell proliferation and mass without negatively affecting function or survival. These findings highlight the future potential therapeutic effectiveness of this peptide under diabetes-related pathophysiological conditions.

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.

Sequential use of Prepidil and extra-amniotic saline infusion for the induction of labor in nulliparous women with very low Bishop scores.

OBJECTIVE: To evaluate the efficacy of sequential use of Prepidil (prostaglandin E2 gel) and extra-amniotic saline infusion for the induction of labor in nulliparous women with very low Bishop scores. STUDY DESIGN: Nulliparous women with singleton gestations, intact membranes and a cervical Bishop score of < or = 2 who received Prepidil gel and extra-amniotic saline infusion sequentially for the induction of labor between July 1996 and July 1998 were studied. RESULTS: Thirty-one women met the inclusion criteria. Indications for induction included post-dates (six of 31), pre-eclampsia (ten of 31), diabetes (three of 31), oligohydramnios (three of 31), intrauterine growth restriction (two of 31) and non-reactive non-stress test (NST) (seven of 31). The average time from onset of induction to delivery was 38.1 +/- 13.5 h. Vaginal delivery was achieved in 80.6%. Women requiring > 2 doses of Prepidil had a higher risk of delivering abdominally (OR = 3.5). Three of seven (42.9%) women with labor induced for non-reactive NST but only three of 24 (12.5%) with labor induced for other indications had a Cesarean section delivery (p < 0.001). CONCLUSIONS: Nulliparous women with very unfavorable cervices can be counselled that they have an 80% chance of vaginal delivery using sequential Prepidil and extra-amniotic saline infusion as an induction method, with 90% delivering within the first 48 h.

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.

Targeted expression of placental lactogen in the beta cells of transgenic mice results in beta cell proliferation, islet mass augmentation, and hypoglycemia.

The factors that regulate pancreatic beta cell proliferation are not well defined. In order to explore the role of murine placental lactogen (PL)-I (mPL-I) in islet mass regulation in vivo, we developed transgenic mice in which mPL-I is targeted to the beta cell using the rat insulin II promoter. Rat insulin II-mPL-I mice displayed both fasting and postprandial hypoglycemia (71 and 105 mg/dl, respectively) as compared with normal mice (92 and 129 mg/dl; p < 0.00005 for both). Plasma insulin concentrations were inappropriately elevated, and insulin content in the pancreas was increased 2-fold. Glucose-stimulated insulin secretion by perifused islets was indistinguishable from controls at 7.5, 15, and 20 mm glucose. Beta cell proliferation rates were twice normal (p = 0. 0005). This hyperplasia, together with a 20% increase in beta cell size, resulted in a 2-fold increase in islet mass (p = 0.0005) and a 1.45-fold increase in islet number (p = 0.0012). In mice, murine PL-I is a potent islet mitogen, is capable of increasing islet mass, and is associated with hypoglycemia over the long term. It can be targeted to the beta cell using standard gene targeting techniques. Potential exists for beta cell engineering using this strategy.

Studies on microencapsulation of 5-fluorouracil with poly(ortho ester) polymers.

Microencapsulation of 5-fluorouracil was successfully accomplished with poly(ortho ester) polymers by the emulsification-solvent evaporation method. While actual drug loading increased with increasing drug load (5-15% w/w), the entrapment efficiency remained essentially unaffected, under a given set of experimental conditions. Incorporation of sorbitan sesquioleate enhanced entrapment efficiency, decreased the volume-surface mean diameter of the poly(ortho ester) microspheres and provided controlled release of 5-fluorouracil. The volume of the aqueous phase was more important than the concentration of polyvinyl alcohol in it. The entrapment efficiency improved from 13 to 33% when the volume of the aqueous phase was increased from 20 to 80 ml. The volume of organic phase (methylene chloride) and the concentration of polymer in it played an important role. The use of smaller volumes of more concentrated polymer solution enhanced actual drug loading, entrapment efficiency and produced larger microspheres. The release studies conducted in 0.01 M phosphate buffer at 37+/-1.0 degrees C demonstrated that the release of 5-FU from the microspheres prepared with sorbitan sequioleate was nearly independent of the initial drug load with a mean zero-order rate constant of 0.0063% per hour. The data suggested that drug release was largely a diffusional process with contributions from dissolution and polymer degradation.

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.

Parathyroid hormone-related protein in the pancreatic islet and the cardiovascular system.

Parathyroid hormone-related protein was discovered as the causative agent responsible for the common paraneoplastic syndrome, humoral hypercalcemia of malignancy. It is now clear that the PTHrP gene is expressed in virtually every cell and tissue in the body at some point in development or adult life and that the peptide is critical for normal life. Two of the tissues that produce PTHrP are the insulin-producing beta cells of the pancreatic islet and the vascular smooth muscle cells of the arterial wall. In this review, the physiologic roles of PTHrP in the islet and in the arterial wall are explored. PTHrP is a classical neuroendocrine prohormone that undergoes extensive post-translational processing to yield a family of daughter peptides that are the mature secretory forms of the peptide. In addition to its ability to act as a traditional endocrine, paracrine, or autocrine factor, PTHrP appears to be able to act as an "intracrine" factor as well, directly entering the nucleus after translation and stimulating proliferation, apoptosis, and perhaps other cellular responses as well. The cell biology underlying this phenomenon is also explored herein.

Progressive pancreatic islet hyperplasia in the islet-targeted, parathyroid hormone-related protein-overexpressing mouse.

PTH-related protein (PTHrP) is a paracrine/autocrine factor produced in most cell types in the body. Its functions include the regulation of cell cycle, of differentiation, of apoptosis, and of developmental events. One of the cells which produces PTHrP is the pancreatic beta cell. We have previously described a transgenic mouse model of targeted overexpression of PTHrP in the beta cell, the RIP-PTHrP mouse. These studies showed that PTHrP overexpression markedly increased islet mass and insulin secretion and resulted in hypoglycemia. Those studies were limited to RIP-PTHrP mice of 8-12 weeks of age. In the current report, we demonstrate that PTHrP overexpression induces a progressive increase in islet mass over the life of the RIP-PTHrP mouse, and that, in contrast to some other models of targeted PTHrP overexpression, the phenotype is not developmental, but occurs postnatally. The marked increase in islet mass is not associated with a measurable increase in beta cell replication rates. A further slowing in the normally low islet apoptosis rate could not be demonstrated in the RIP-PTHrP islet. Thus, the marked increase in islet mass in the RIP-PTHrP mouse is unexplained in mechanistic terms. Finally, RIP-PTHrP mice are resistant to the diabetogenic effects of streptozotocin. The mechanisms responsible for the increase in islet mass in the RIP-PTHrP mouse likely lie in either very subtle changes in islet turnover or in early steps in islet differentiation and development. The ability of PTHrP to increase islet mass and function, as well as its ability to attenuate the diabetogenic effects of streptozotocin, indicate that further study of PTHrP on islet development and function are important and may lead to therapeutic strategies in diabetes mellitus.

Transfection and overexpression of the calcium binding protein calbindin-D28k results in a stimulatory effect on insulin synthesis in a rat beta cell line (RIN 1046-38).

Calbindin-D28k, a calcium binding protein that is thought to act as a facilitator of calcium diffusion in intestine and kidney, is known to be regulated by vitamin D in these tissues. Calbindin-D28k is also present in pancreatic beta cells, but its function in these cells is not known. To determine a role for calbindin-D28k in the beta cell, rat calbindin-D28k was overexpressed in the pancreatic beta cell line RIN 1046-38 by transfection of calbindin in expression vector, and changes in insulin mRNA were examined. Five transfected RIN cell clones were found to overexpress calbindin 6- to 35-fold as determined by radioimmunoassay. Northern blot analysis revealed increases in abundance in calbindin mRNA (>20-fold for most clones). Overexpressed calbindin was functional because it was capable of buffering calcium in response to a rapid calcium influx induced by 1 and 5 microM calcium ionophore. In cells transfected with calbindin, there was a marked increase in the expression of insulin mRNA (>20-fold for most clones compared with vector transfected cells). Besides an increase in insulin mRNA, calbindin overexpression was also associated with an increase in insulin content and release (a 5.8-fold increase in insulin release was noted for clone C10, and a 54-fold increase was noted for clone C2). To begin to address the mechanism whereby overexpression of calbindin results in increased insulin gene expression, calbindin-overexpressing clones were transiently transfected with plasmids incorporating various regions of the rat insulin I (rInsI) promoter linked to the chloramphenicol acetyltransferase coding sequence. Transient transfection with reporter plasmids bearing the regulatory sequences of the rInsI promoter (-345/+1) or five copies of the Far-FLAT minienhancer (-247/-198) from the rInsI promoter suggests that increased insulin mRNA in calbindin transfected cells is due, at least in part, to enhanced insulin gene transcription. These studies provide the first direct evidence (to our knowledge) for a role for calbindin in beta cell function.

Opposing mitogenic and anti-mitogenic actions of parathyroid hormone-related protein in vascular smooth muscle cells: a critical role for nuclear targeting.

Parathyroid hormone-related protein (PTHrP) is a prohormone that is posttranslationally processed to a family of mature secretory forms, each of which has its own cognate receptor(s) on the cell surface that mediate the actions of PTHrP. In addition to being secreted via the classical secretory pathway and interacting with cell surface receptors in a paracrine/autocrine fashion, PTHrP appears to be able to enter the nucleus directly following translation and influence cellular events in an "intracrine" fashion. In this report, we demonstrate that PTHrP can be targeted to the nucleus in vascular smooth muscle cells, that this nuclear targeting is associated with a striking increase in mitogenesis, that this nuclear effect on proliferation is the diametric opposite of the effects of PTHrP resulting from interaction with cell surface receptors on vascular smooth muscle cells, and that the regions of the PTHrP sequence responsible for this nuclear targeting represent a classical bipartite nuclear localization signal. This report describes the activation of the cell cycle in association with nuclear localization of PTHrP in any cell type. These findings have important implications for the normal physiology of PTHrP in the many tissues which produce it, and suggest that gene delivery of PTHrP or modified variants may be useful in the management of atherosclerotic vascular disease.

Structural and physiologic characterization of the mid-region secretory species of parathyroid hormone-related protein.

Parathyroid hormone-related protein (PTHrP) is initially translated as a preprohormone which is posttranslationally processed to yield a family of mature secretory forms. Most attention has focused on the amino-terminal portion of the molecule which is homologous to parathyroid hormone. It is clear, however, that a mid-region species of PTHrP is posttranslationally cleaved from the highly conserved mid-region of PTHrP, and that the amino terminus of this peptide is Ala38. The purposes of the current study were three: 1) to confirm that Arg37 immediately preceding Ala38 serves as a posttranslational processing site in the PTHrP precursor, 2) to determine the carboxyl terminus of the mid-region secretory species of PTHrP, and 3) to synthesize this authentic mid-region secretory form of PTHrP and determine whether it is biologically active. The results indicate that: 1) Arg37 is indeed a processing site in the PTHrP precursor; 2) three distinct mid-region PTHrP species are generated by posttranslational processing, PTHrP(38-94)amide, PTHrP(38-95), and most likely, PTHrP(38-101); and 3) synthetic mid-region PTHrP(38-94)amide is active in four different biological systems. These studies confirm the finding that PTHrP is a prohormone. More importantly, they define a novel, biologically active highly conserved mid-region secretory form of PTHrP.

Transactivation of the PTHrP gene in squamous carcinomas predicts the occurrence of hypercalcemia in athymic mice.

Humoral hypercalcemia of malignancy (HHM) is caused by the secretion of parathyroid hormone-related protein (PTHrP) by tumor cells, and tumors of squamous histology are the ones most commonly complicated by HHM. To determine why some squamous tumors cause HHM and others do not, we quantitated the levels of PTHrP mRNA expression and PTHrP secretion in a series of eight squamous tumor lines. As anticipated, we found that the level of PTHrP mRNA expression in individual lines correlated with their PTHrP secretion rates. However, PTHrP mRNA levels varied widely in individual lines, and only those tumor lines with the highest levels of PTHrP gene expression were able to cause hypercalcemia in athymic mice. We found that a specific segment of the PTHrP promoter could reproduce the relative pattern of PTHrP gene expression when cloned in front of a chloramphenicol acetyltransferase reporter gene and transiently transfected into these squamous lines. Deletional analysis confirmed that specific sequences within the PTHrP gene promoter appeared to be involved in the transactivation of the gene in tumor lines expressing high levels of PTHrP mRNA. These data suggest that the ability of a given squamous tumor to cause HHM is ultimately a function of its level of PTHrP gene expression, which in turn appears to be a function of the ability of specific transcription factors to transactivate PTHrP gene expression.

Overexpression of parathyroid hormone-related protein in the pancreatic islets of transgenic mice causes islet hyperplasia, hyperinsulinemia, and hypoglycemia.

Parathyroid hormone-related protein (PTHrP) is produced by the pancreatic islet. It also has receptors on islet cells, suggesting that it may serve a paracrine or autocrine role within the islet. We have developed transgenic mice, which overexpress PTHrP in the islet through the use of the rat insulin II promoter (RIP). Glucose homeostasis in these mice is markedly abnormal; RIP-PTHrP mice are hypoglycemic in the postprandial and fasting states and display inappropriate hyperinsulinemia. At the end of a 24-hour fast, blood glucose values are 49 mg/dl in RIP-PTHrP mice, as compared to 77 mg/dl in normal littermates; insulin concentrations at this time are 6.3 and 3.9 ng/ml, respectively. Islet perifusion studies failed to demonstrate abnormalities in insulin secretion. In contrast, quantitative islet histomorphometry demonstrates that the total islet number and total islet mass are 2-fold higher in RIP-PTHrP mice than in their normal littermates. PTHrP very likely plays a normal physiologic role within the pancreatic islet. This role is most likely paracrine or autocrine. PTHrP appears to regulate insulin secretion either directly or indirectly, through developmental or growth effects on islet mass. PTHrP may have a role as an agent that enhances islet mass and/or enhances insulin secretion.

Defining the roles of parathyroid hormone-related protein in normal physiology.

Parathyroid hormone-related protein (PTHrP) was discovered as a result of a search for the circulating factor secreted by cancers which causes the common paraneoplastic syndrome humoral hypercalcemia of malignancy. Since the identification of the peptide in 1982 and the cloning of the cDNA in 1987, it has become clear that PTHrP is a prohormone that is posttranslationally cleaved by prohormone convertases to yield a complex family of peptides, each of which is believed to have its own receptor. It is also clear that the PTHrP gene is expressed not only in cancers but also in the vast majority of normal tissues during adult and/or fetal life. In contrast to the situation in humoral hypercalcemia of malignancy in which PTHrP plays the role of a classical "endocrine" hormone, under normal circumstances PTHrP plays predominantly paracrine and/or autocrine roles. These apparent physiological functions are also complex and appear to include 1) regulation of smooth muscle (vascular, intestinal, uterine, bladder) tone, 2) regulation of transepithelial (renal, placental, oviduct, mammary gland) calcium transport, and 3) regulation of tissue and organ development, differentiation, and proliferation. In this review, the discovery of PTHrP, the structure of its gene and its cDNAs, and the posttranslational processing of the initial translation products are briefly reviewed. Attention is then focused on a detailed organ system-oriented review of the normal physiological functions of PTHrP.

Further evidence for a novel receptor for amino-terminal parathyroid hormone-related protein on keratinocytes and squamous carcinoma cell lines.

PTH and PTH-related peptides (PTHrPs) interact with a common PTH/PTHrP receptor (type I), which is expressed in many tissues, including bone and kidney. Amino-terminal PTH and PTHrPs also recognize receptors in several nonclassical PTH target tissues, and in some of these, the signaling mechanisms differ qualitatively from those of the classical type I receptor. In normal keratinocytes and squamous carcinoma cell lines, PTH and PTHrP stimulate a rise in intracellular calcium, but not cAMP, suggesting the existence of an alternate, type II PTH/PTHrP receptor. SqCC/Y1 squamous carcinoma cells stably expressing the type I receptor displayed sensitive intracellular cAMP responses to PTHrP and PTH, indicating that these cells express functional GS proteins and that the type I receptor is capable of signaling through adenylyl cyclase in this cell line. Therefore, the endogenous type II receptor in SqCC/Y1 cells differs from the cloned type I receptor. We next examined whether messenger RNA (mRNA) from keratinocytes and squamous cell lines could hybridize to a human type I PTH/PTHrP receptor complementary DNA [1.9 kilobases (kb)]. No type I receptor mRNA (2.3 kb) was detected in polyadenylated RNA from any of the squamous cell lines. However, squamous cell lines did express several mRNA transcripts that hybridized with the type I receptor probe, yet were smaller (1 and 1.5 kb) or larger (3.5-5 kb) than the cloned receptor mRNA. The predominant mRNA in two squamous carcinoma cell lines and normal keratinocytes was a 1-kb transcript. Northern analysis with five different region-specific probes that span the entire coding region of the human type I receptor was used to map homologous regions within each of the transcripts. Several of the transcripts identified in squamous lines are also present in polyadenylated RNA from SaOS-2 human bone cells, but a unique 1-kb transcript hybridizing to probe 2 (nucleotides 490-870) was observed only in squamous cells. The smaller 1- and 1.5-kb transcripts did not hybridize to probes corresponding to the extreme 5'- and 3'-coding regions of the type I receptor complementary DNA. Ribonuclease protection analysis employing riboprobes that correspond to the five region-specific DNA probes revealed strong RNA signals of the expected size in SaOS-2 cells, but no hybridization with squamous cell RNA. Several smaller, but minor, bands that were unique to squamous cells were observed with riboprobe 2 only, suggesting partial homology of this region with the type I receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Region-specific methylation of the parathyroid hormone-related peptide gene determines its expression in human renal carcinoma cell lines.

Tumor production of a parathyroid hormone-related peptide (PTHrP) is a common cause of the syndrome of humoral hypercalcemia of malignancy, which is frequently associated with renal cell carcinomas. Why certain renal cell carcinomas produce PTHrP while others do not is unknown. Using a system of 12 human renal carcinoma cell lines which either do (n = 6) or do not (n = 6) produce PTHrP, we found that the expression of the PTHrP gene in these cell lines is controlled at the transcriptional level. Transfection studies failed to demonstrate variation in PTHrP promoter activity in these cell lines sufficient to account for the differential PTHrP expression, implicating a cis-acting mechanism. Transcription of the PTHrP gene in these cell lines was found to correlate with the methylation state of specific CpG dinucleotides located within the promoter region but outside a CpG island. The functional importance of this mechanism of control was confirmed by the ability of the demethylating agent, 5-azacytidine, to induce PTHrP mRNA expression in previously nonexpressing cell lines.