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.

Glucose stimulates human beta cell replication in vivo in islets transplanted into NOD-severe combined immunodeficiency (SCID) mice.

AIMS/HYPOTHESIS: We determined whether hyperglycaemia stimulates human beta cell replication in vivo in an islet transplant model METHODS: Human islets were transplanted into streptozotocin-induced diabetic NOD-severe combined immunodeficiency mice. Blood glucose was measured serially during a 2 week graft revascularisation period. Engrafted mice were then catheterised in the femoral artery and vein, and infused intravenously with BrdU for 4 days to label replicating beta cells. Mice with restored normoglycaemia were co-infused with either 0.9% (wt/vol.) saline or 50% (wt/vol.) glucose to generate glycaemic differences among grafts from the same donors. During infusions, blood glucose was measured daily. After infusion, human beta cell replication and apoptosis were measured in graft sections using immunofluorescence for insulin, and BrdU or TUNEL. RESULTS: Human islet grafts corrected diabetes in the majority of cases. Among grafts from the same donor, human beta cell proliferation doubled in those exposed to higher glucose relative to lower glucose. Across the entire cohort of grafts, higher blood glucose was strongly correlated with increased beta cell replication. Beta cell replication rates were unrelated to circulating human insulin levels or donor age, but tended to correlate with donor BMI. Beta cell TUNEL reactivity was not measurably increased in grafts exposed to elevated blood glucose. CONCLUSIONS/INTERPRETATION: Glucose is a mitogenic stimulus for transplanted human beta cells in vivo. Investigating the underlying pathways may point to mechanisms capable of expanding human beta cell mass in vivo.

Deficiency of Atf3, an adaptive-response gene, protects islets and ameliorates inflammation in a syngeneic mouse transplantation model.

AIMS/HYPOTHESIS: Islet transplantation is a potential therapeutic option for type 1 diabetes. However, the need for multiple donors per patient and heavy immunosuppression of the recipients limit its use. The goal of this study was to test whether the gene encoding activating transcription factor 3 (ATF3), a stress-inducible pro-apoptotic gene, plays a role in graft rejection in islet transplantation. METHODS: We compared wild-type (WT) and Atf3 knockout (KO) islets in vitro using stress paradigms relevant to islet transplantation: isolation, inflammation and hypoxia. We also compared the WT and KO islets in vivo using a syngeneic mouse transplantation model. RESULTS: ATF3 was induced in all three stress paradigms and played a deleterious role in islet survival, as evidenced by the lower viability of WT islets compared with KO islets. ATF3 upregulated various downstream target genes in a stress-dependent manner. These target genes can be classified into two functional groups: (1) apoptosis (Noxa [also known as Pmaip1] and Bnip3), and (2) immunomodulation (Tnfalpha [also known as Tnf], Il-1beta [also known as Il1b], Il-6 [also known as Il6] and Ccl2 [also known as Mcp-1]). In vivo, Atf3 KO islets performed better than WT islets after transplantation, as evidenced by better glucose homeostasis in the recipients and the reduction of the following variables in the KO grafts: caspase 3 activation, macrophage infiltration and expression of the above apoptotic and immunomodulatory genes. CONCLUSIONS/INTERPRETATION: ATF3 plays a role in islet graft rejection by contributing to islet cell death and inflammatory responses at the graft sites. Silencing the ATF3 gene may provide therapeutic benefits in islet transplantation.

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.

Parathyroid hormone-related protein is an autocrine modulator of rabbit proximal tubule cell growth.

Parathyroid hormone-related protein (PTHrP), a likely mediator for humoral hypercalcemia of malignancy, is also synthesized in various normal tissues. In the kidney, PTHrP, mainly detected in proximal and distal tubules, has been shown to stimulate proliferation of rat mesangial cells in culture. Experiments were carried out to investigate the possible mitogenic effect of PTHrP in cultures of rabbit proximal tubule cells (PTC). Immunocytochemical analysis, using antihuman (h)PTHrP antibodies to (38-64) and (107-111) epitopes in the PTHrP molecule, showed strong cytoplasmic staining in PTC and proximal tubule-like LLC-PK1 cells. PTC secreted immunoreactive PTHrP (54.8 +/- 7.0 fmol/10(6) cells) into the culture medium. Human PTHrP(1-141) stimulated proliferation in subconfluent cultures of these cells dose-dependently. This effect was similar to that induced by [Tyr34]hPTHrP(1-34) amide (hPTHrP[1-34]), hPTHrP(1-86), and bovine (b)PTH(1-34), while hPTHrP(38-64) amide, hPTHrP9107-111) amide, and hPTHrP(107-139) amide were ineffective. Addition of anti-hPTHrP neutralizing antibodies to (1-34), (38-64), and (107-111) epitopes of PTHrP decreased PTC growth. The mitogenic effect of these agonists was abolished in confluent PTC. In contrast, [Nle8,18, Tyr34]bPTH(3-34)amide (bPTH[3-34]) increased DNA synthesis in either subconfluent or confluent PTC. In LLC-PK1 cells, which also secreted PTHrP and are devoid of PTH receptors, none of these peptides affected proliferation. Forskolin (10 microM) or H-8 (2 microM), a protein kinase A inhibitor, did not affect basal or hPTHrP(1-34)-stimulated DNA synthesis, respectively, in subconfluent PTC. On the other hand, 10 nM staurosporine and 100 nM calphostin C, protein kinase C (PKC) inhibitors, blunted the effects of hPTHrP(1-34) or bPTH(3-34) on DNA synthesis in these cells. These studies suggest that PTHrP may function as an autocrine factor in the regulation of proximal tubule cell growth by a PKC-mediated mechanism.

C-terminal parathyroid hormone-related protein inhibits proliferation and differentiation of human osteoblast-like cells.

Parathyroid hormone-related protein (PTHrP) is synthesized by osteoblasts, although its local role in bone is not completely understood. The C-terminal (107-111) region of PTHrP seems to be a potent inhibitor of osteoblastic bone resorption. We studied the effect of this PTHrP domain on the proliferation and synthesis of osteoblastic markers in osteoblast-like cells from adult human bone. We found that the human (h)PTHrP(107-139) fragment, between 10 fM and 10 nM, inhibited 3H-thymidine incorporation into these cells. The antiproliferative effect of the latter fragment, or that of hPTHrP(107-111), was similar to that induced by [Tyr34] hPTHrP(1-34) amide, bovine PTH(1-34), and hPTHrP(1-141), while hPTHrP(38-64) amide was ineffective. Human PTHrP(7-34) amide, at 10 nM, and 1 microM phorbol-12-myristate-13-acetate also significantly decreased DNA synthesis in human osteoblast-like cells. Neither hPTHrP(7-34) amide nor hPTHrP(107-139), at 10 nM, stimulated protein kinase A (PKA) activity in these cells. Moreover, 100 nM H-89, a PKA inhibitor, did not eliminate the inhibitory effect of hPTHrP(107-139) on these cells' growth. However 100 nM calphostin C, a PKC inhibitor, blunted this effect of PTHrP(107-139). In addition to their antimitogenic effect, hPTHrP(107-139) and hPTHrP(107-111) inhibited basal and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-stimulated alkaline phosphatase activity in these cells. Both fragments, like 1,25(OH)2D3, decreased C-terminal type I procollagen secretion into the cell-conditioned medium, but osteocalcin secretion by these cells was unaffected by the C-terminal PTHrP fragments. These findings suggest that PTHrP may act as a local regulator of bone formation.

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.

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.

Parathyroid hormone-related protein-(1--36) stimulates renal tubular calcium reabsorption in normal human volunteers: implications for the pathogenesis of humoral hypercalcemia of malignancy.

All would agree that hypercalcemia occurs among patients with humoral hypercalcemia of malignancy (HHM) as a result of osteoclastic bone resorption. Some studies suggest that enhanced renal calcium reabsorption, which plays an important pathophysiological role in the hypercalcemia occurring in primary hyperparathyroidism, is also important pathophysiologically in HHM. Other studies have not agreed. In large part, these differences result from the inability to accurately assess creatinine and calcium clearance in critically ill subjects with HHM. To circumvent these issues, we have developed steady state 48-h PTH-related protein (PTHrP) infusion and 8-h hypercalcemic calcium clamp protocols. These techniques allow assessment of the effects of steady state PTHrP and calcium infusions in normal healthy volunteers in a setting in which renal function is stable and measurable and in which the filtered load of calcium can be matched in PTHrP- and calcium-infused subjects. Normal subjects were infused with saline (placebo), PTHrP, or calcium. Subjects receiving PTHrP, as expected, displayed mild hypercalcemia (10.2 mg/dL), suppression of endogenous PTH-(1--84), and phosphaturia. Subjects receiving the hypercalcemic calcium clamp displayed indistinguishable degrees of hypercalcemia and PTH suppression. Despite their matched degrees of hypercalcemia and PTH suppression, the two groups differed importantly with regard to fractional calcium excretion (FECa). The hypercalcemic calcium clamp group was markedly hypercalciuric (FECa averaged 6.5%), whereas FECa in the PTHrP-infused subjects was approximately 50% lower (between 2.5--3.7%), and no different from that in the normal controls, which ranged from 1.5--3.0%. These studies demonstrate that PTHrP is able to stimulate renal calcium reabsorption in healthy volunteers. These studies suggest that PTHrP-induced renal calcium reabsorption, in concert with the well established acceleration of osteoclastic bone resorption, contributes in a significant way to the hypercalcemia observed in patients with HHM.

Cyclosporine increases renal parathyroid hormone-related protein expression in vivo in the rat.

BACKGROUND: Clinical use of cyclosporine (CsA) is limited by its known nephrotoxicity. Parathyroid hormone (PTH)-related protein (PTHrP) increases after acute renal ischemia and stimulates proliferation of renal cells in culture. Herein, we have examined whether the renal expression of PTHrP and its PTH/PTHrP receptor is affected by chronic CsA nephrotoxicity. METHODS: Rats were randomly assigned to receive daily intramuscular injections of either CsA (25 mg/kg) or the same volume of the vehicle olive oil (control) for 3 weeks. At this time interval, under ether anesthesia, rat blood and kidneys were obtained for analytical determinations, and total RNA isolation or immunohistochemistry, respectively. RESULTS: Serum urea was 11+/-2 and 6+/-1 mmol/L (P < 0.01) in CsA-treated and control rats, respectively. We found that PTH/PTHrP receptor mRNA was unchanged, but PTHrP mRNA, and also transforming growth factor-beta1 mRNA expression as positive control, was about twofold increased in the kidney of CsA-treated rats. This was accompanied by increased PTHrP immunostaining in renal cortical tubules, associated with tubule vacuolation. CONCLUSION: This study demonstrates an up-regulation of PTHrP, associated with chronic CsA-induced nephrotoxicity. Our findings support a role for PTHrP in the CsA-injured kidney.

Characterization of parathyroid hormone/parathyroid hormone-related protein receptor and signaling in hypercalcemic Walker 256 tumor cells.

Parathyroid hormone (PTH)-related protein (PTHrP) is the main factor responsible for humoral hypercalcemia of malignancy. Both PTH and PTHrP bind to the common type I PTH/PTHrP receptor (PTHR), thereby activating phospholipase C and adenylate cyclase through various G proteins, in bone and renal cells. However, various normal and transformed cell types, including hypercalcemic Walker 256 (W256) tumor cells, do not produce cAMP after PTHrP stimulation. We characterized the PTHrP receptor and the signaling mechanism upon its activation in the latter cells. Scatchard analysis of PTHrP-binding data in W256 tumor cells revealed the presence of high affinity binding sites with an apparent K(d) of 17 nM, and a density of 90 000 sites/cell. In addition, W256 tumor cells immunostained with an anti-PTHR antibody, recognizing its extracellular domain. Furthermore, reverse transcription followed by PCR, using primers amplifying two different regions in the PTHR cDNA corresponding to the N- and C-terminal domains, yielded products from W256 tumor cell RNA which were identical to the corresponding products obtained from rat kidney RNA. Consistent with our previous findings on cAMP production, 1 microM PTHrP(1-34), in contrast to 10 microg/ml cholera toxin or 1 microM isoproterenol, failed to affect protein kinase A activity in W256 tumor cells. However, in these cells we found a functional PTHR coupling to G(alpha)(q/11), whose presence was demonstrated in these tumor cell membranes by Western blot analysis. Our findings indicate that W256 tumor cells express the PTHR, which seems to be coupled to G(alpha)(q/11). Taken together with previous data, these results support the hypothesis that a switch from the cAMP pathway to the phospholipase C-intracellular calcium pathway, associated with PTHR activation, occurs in malignant cells.

Comparison of antiproliferative effects of atrial natriuretic peptide and transforming growth factor beta on rabbit kidney proximal tubule cells.

The effects of atrial natriuretic peptide (ANP) and transforming growth factor beta (TGF beta) on cell proliferation were investigated in rabbit kidney proximal tubule cells. At 48 h, each agonist inhibited cell growth dose dependently. Moreover, the antiproliferative effect of both factors together was greater than that of each factor alone. However, coincubation of a high concentration (100 nM) of ANP and epidermal growth factor (EGF) was found to induce cell hypertrophy. This hypertrophic effect induced by ANP in the presence of EGF was mimicked by 8-BrcGMP but not by various cAMP analogues, at 100 microM, and was independent of the tyrosine kinase inhibitor genistein (10 microM). However, it was inhibited by 100 pM TGF beta. These in vitro results suggest that the antiproliferative effects of ANP and TGF beta may be part of a mechanism to modulate the hyperplastic effect of EGF in the renal proximal tubule during compensatory kidney growth.

Parathyroid hormone-related protein increases DNA synthesis in proximal tubule cells by cyclic AMP- and protein kinase C-dependent pathways.

The present study was performed to characterize the possible involvement of cAMP synthesis and protein kinase C (PKC) activation in the DNA synthesis-stimulating effect of parathyroid hormone-related protein (PTHrP) in proximal tubule cells. We found that DNA synthesis was stimulated by 10 microM 8BrcAMP, and 1 microM Sp-cDBIMPS, two cAMP analogs, and also by 1 microM phorbol 12-myristate 13-acetate (PMA) and 100 microM 1,2-dioctanoyl-sn-glycerol, two PKC activators, and 10 nM [Cys23] human (h)PTHrP (24-35) amide in rabbit proximal tubule cells (PTC). Both Sp-cDBIMPS and PMA, at 1 microM, also increased DNA synthesis in SV40-immortalized mouse proximal tubule cells MCT. Human PTHrP (7-34) amide [PTHrP (7-34)] dose dependently stimulated DNA synthesis in a similar manner as [34Tyr]PTHrP (1-34) amide [PTHrP (1-34)], in PTC. PMA pre-treatment for 20 h, which downregulates PKC, completely blocked the effect induced by PTHrP (7-34), but not that of PTHrP (1-34), in the latter cells. In contrast, the same PMA pre-treatment abolished the DNA synthesis stimulation by PTHrP (1-34) and PTHrP (7-34) in MCT cells, which appear to have PTH receptors mainly coupled to phospholipase C and not adenylate cyclase. Our results indicate that the stimulatory effect of PTHrP on DNA synthesis in proximal tubule cells is mediated by a cAMP- and PKC-dependent mechanism.

Hypertrophy of rabbit proximal tubule cells is associated with overexpression of TGF beta.

The expression of transforming growth factor beta (TGF beta) in proximal tubule cells from rabbit kidney cortex after uninephrectomy (UNX) was investigated. Cell protein and [3H]leucine incorporation in these cells were significantly increased, while cell number was decreased, at two weeks following UNX. At this time period after UNX, we found that proximal tubule cells showed a dramatic increase of cytoplasmic immunostaining with a pan-specific anti-TGF beta antibody. This was accompanied by a 3-fold increase in TGF beta 1 mRNA expression in these cells. Furthermore, proximal tubule cells from two-week uninephrectomized rabbits secrete about 2-fold higher TGF beta bioactivity to the cell conditioned medium compared to cells from sham-operated animals. Addition of anti-TGF beta 1, beta 2, beta 3 neutralizing antibody increased the growth of the former cells, and it abolished cell hypertrophy. These results indicate that hypertrophy of proximal tubule cells during compensatory renal growth is associated with overexpression of TGF beta.

Functional roles of the nuclear localization signal of parathyroid hormone-related protein (PTHrP) in osteoblastic cells.

PTHrP is an important regulator of bone remodelling, apparently by acting through several sequence domains. We here aimed to further delineate the functional roles of the nuclear localization signal (NLS) comprising the 88-107 amino acid sequence of PTHrP in osteoblasts. PTHrP mutants from a human PTHrP (-36/+139) cDNA (wild type) cloned into pcDNA3.1 plasmid with deletion (Δ) of the signal peptide (SP), NLS, T(107), or T107A replacing T(107) by A(107) were generated and stably transfected into osteoblastic MC3T3-E1 cells. In these cells, intracellular trafficking, cell proliferation and viability, as well as cell differentiation were evaluated. In these transfected cells, PTHrP was detected in the cytoplasm and also in the nucleus, except in the NLS mutant. Meanwhile, the PTH type 1 receptor (PTH1R) accumulates in the cytoplasm except for the ΔSP mutant in which the receptor remains at the cell membrane. PTHrP-wild type cells showed enhanced growth and viability, as well as an increased matrix mineralization, alkaline phosphatase activity, and osteocalcin gene expression; and these features were inhibited or abolished in ΔNLS or ΔT(107) mutants. Of note, these effects of PTHrP overexpression on cell growth and function were similarly decreased in the ΔSP mutant after PTH1R small interfering RNA transfection or by a PTH1R antagonist. The present in vitro findings suggest a mixed model for PTHrP actions on osteoblastic growth and function whereby this protein needs to be secreted and internalized via the PTH1R (autocrine/paracrine pathway) before NLS-dependent shuttling to the nucleus (intracrine pathway).

Role of kidney and liver in the renotropic activity generated in rats after uninephrectomy.

Following uninephrectomy the remnant kidney undergoes a compensatory growth apparently regulated by a humoral renotropic factor(s). Our studies were carried out to assess the role of renal and hepatic tissue in the generation of such a renotropic factor(s). The renotropic activity in extracts of rat kidney and liver obtained at different times after uninephrectomy was assayed in cortical tubules from rat kidney removed 24 h after uninephrectomy. We found that kidney extracts from 6 h and 24 h uninephrectomized rats increased [3H]thymidine incorporation into tubular cell DNA, dose-dependently, compared to those from sham-operated rats. Maximal stimulation was observed at 10(4)-fold (6 h) or 10(2)-fold (24 h) extract dilution. This activity was undetected in perfused kidney extracts, or in extracts of kidneys from simultaneously uninephrectomized and partially hepatectomized rats. However, only 10-fold dilution of unperfused liver extract from 6 h or 24 h uninephrectomized rats showed significant activity. Kidney or liver extracts from 30 min uninephrectomized animals failed to display activity. Our results point to an extrarenal origin for the humoral renotropic activity generated after uninephrectomy, although the kidney seems to modulate this activity.

Further studies on the characterization of a renotropic activity detected in uninephrectomized rat plasma.

Using chromatographic techniques, we have purified a peptide responsible, at least in part, for the renotropic activity detected in 24-hour uninephrectomized adult rat plasma. The most purified material behaved as a single component when analyzed by reversed-phase HPLC. The purified factor stimulates DNA synthesis in isolated rat proximal tubules and proximal tubular cell (LLC-PK1) cultures. This effect was abolished by pretreatment with protease K, with an intracellular calcium chelator or with indomethacin. This factor was also mitogenic for rat mesangial cells. In contrast, neither Madin-Darby canine kidney cells nor rat liver cells responded to the growth factor. Using the same purification procedure, a bioactive factor with an amino acid composition almost identical to that of this renotropic peptide was isolated from sham-operated or normal rat plasma. However, only uninephrectomized plasma after gel filtration showed renotropic activity, which was inhibited by the corresponding chromatographic fraction from sham-operated plasma. Our findings suggest that a change in the concentration of a circulating inhibitor(s) appears to account for the differences in the activity of sham-operated or control plasma and uninephrectomized plasma on the growth of renal cells.

Transforming growth factor-beta and its receptors in rabbit renal proximal tubules after uninephrectomy.

Transforming growth factor-beta (TGF beta) and its receptors were studied in proximal tubules isolated from rabbit renal cortex at different times after uninephrectomy (UNX). Scatchard analysis of TGF beta-binding data in proximal tubules from control kidneys revealed two types of binding sites, with Kd 21 and 208 pM, and Bmax 33 and 104 fmol/mg protein, respectively. Kd values were similar in these animals at 1 or 2 weeks after either UNX or sham operation (SNX). However, Bmax increased in parallel with the observed increase in the protein/DNA ratio of the proximal tubules at 2 weeks after UNX. In contrast, [125I]-insulin binding per milligram of protein was lower in proximal tubules from uninephrectomized compared to sham-operated animals within the same time period. Affinity labeling of [125I]-TGF beta 1-binding sites in proximal tubules from either sham-operated or uninephrectomized rabbits displayed two labeled proteins with apparent molecular weights of > 143 and 43 kD. We found an increased TGF beta bioactivity in the conditioned medium of proximal tubules at 2 weeks following UNX. This protein increase was associated with an increased TGF beta 1 mRNA expression in these tubules. In contrast, no significant changes in TGF beta bioactivity were observed in rabbit glomeruli conditioned medium or in rabbit urine at this time period after UNX. Our data indicate that hypertrophy of the proximal tubule is associated with an increased TGF beta production and a lack of downregulation of its receptors in this nephron portion.

Partial purification and characterisation of a renal growth factor from plasma of uninephrectomised rats.

Renal growth factor activity was extracted from plasma of adult uninephrectomised rats and partially purified by gel filtration and anion-exchanger FPLC. It induced a maximal stimulation of mouse DNA synthesis in vivo at 1.75 micrograms/mouse. In addition, renal growth factor was found to maximally stimulate DNA synthesis in LLC-PK1 cells at 150 ng/ml. This maximal response was then found to decrease with higher doses of renal growth factor, in vivo and in vitro. The apparent molecular weight of renal growth factor was estimated to be 17K-22 K by gel filtration. It was found to be resistant to heat and to trypsin, but labile to reduction with dithiothreitol.