Parathyroidectomy does not prevent the renal PTH/PTHrP receptor down-regulation in uremic rats

In a recent study we demonstrated that the PTH/PTHrP receptor (PTH-R) mRNA was markedly down-regulated in the remnant kidney of uremic rats with severe secondary hyperparathyroidism. Among the factors potentially implicated in this down-regulation, to date only PTH has been demonstrated to modulate PTH-R expression. Here, we examined the effect of thyroparathyroidectomy (TPTX) on the renal expression of PTH-R in rats with normal renal function or with chronic renal failure (CRF) induced by 5/6 nephrectomy. Four groups of rats were studied: control, TPTX, CRF, and CRF + TPTX. Moderate-degree renal failure was documented by mean (+/- SD) creatinine clearances (microliter/min/100 g body wt) of 259 +/- 40 and 212 +/- 45 in CRF and CRF + TPTX rats, compared with 646 +/- 123 and 511 +/- 156 in control and TPTX rats, respectively. Plasma phosphorus, calcitriol, and ionized calcium were significantly lower in CRF and CRF + TPTX than in control animals. Plasma ionized calcium and calcitriol were also lower in TPTX than in control rats. Plasma PTH levels (pg/ml) were increased in CRF rats (41.8 +/- 29.4), and markedly decreased in TPTX (10.1 +/- 7.8) and CRF + TPTX (8.0 +/- 3.8) rats compared with control rats (21.7 +/- 7.5). Northern blot analysis showed that the level of the steady-state PTH-R mRNA in the kidney of CRF and CRF + TPTX rats was markedly decreased compared with that of control rats, the ratios of PTH-R mRNA/beta-actin mRNA being 0.28 +/- 0.04 and 0.27 +/- 0.03 versus 0.54 +/- 0.05, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parathyroid hormone-related peptide (PTHrP) and parathyroid hormone (PTH)/PTHrP receptor

Parathyroid hormone-related peptide (PTHrP) has been identified as the factor responsible for the humoral hypercalcemia of malignancy (HHM). Since the cloning of the cDNA, it has become clear that PTHrP is a prohormone that is posttranslationally cleaved to yield a complex family of peptides. Through its homology to parathyroid hormone (PTH) in the amino-terminus region of the protein, it is able to bind to and activate a common PTH/PTHrP receptor. PTHrP has been shown to be a normal product of many adult and fetal tissues, where it appears to act in an autocrine/paracrine fashion to regulate organogenesis. PTHrP and the PTH/PTHrP receptor seem to be co-expressed in many tissues, but their role in the various systems is uncertain. The use of transgenic and knock-out animal models has contributed to a better understanding of the physiological role of this peptide and its receptor. In this review, the structure of their genes, their expression pattern, and some of their major physiological functions are discussed. Attention is focused on their interaction in the regulation of cartilage and bone development.     

Conditionally immortalized murine bone marrow stromal cells mediate parathyroid hormone-dependent osteoclastogenesis in vitro

PTH recruits and activates osteoclasts to cause bone resorption. These actions of PTH are thought to be mediated indirectly via type 1 PTH/PTH-related peptide receptors (PTH1Rs) expressed by adjacent marrow stromal or osteoblastic cells, although some evidence suggests that PTH may act directly on early hematopoietic osteoclast progenitors. We have established clonal, conditionally immortalized, PTH-responsive, bone marrow stromal cell lines from mice that harbor both a transgene encoding a temperature-sensitive mutant of the simian virus 40 large T antigen and deletion of a single allele of the PTH1R gene. Of 60 stromal cell lines isolated, 45 expressed functional PTH1Rs. During coculture with normal murine spleen cells, 5 of 42 such cell lines could support formation of tartrate-resistant acid phosphatase-positive, multinucleated cells (TRAP+ MNCs) in response to 1,25-dihydroxyvitamin D3, but only 2 of these did so in response to PTH. One of these, MS1 cells, expressed numerous cytokines and proteins characteristic of the osteogenic lineage and showed increased production of interleukin-6 in response to PTH. MS1 cells supported dose-dependent induction by rat (r) PTH-(1-34) (0.1-100 nM) of TRAP+ MNCs that expressed calcitonin receptors and formed resorption lacunae on dentine slices. This effect of PTH, which required cell to cell contact between MS1 and spleen cells, was mimicked by coadministration of cAMP analog and phorbol ester but only partially by either agent alone. The carboxyl-terminal fragment rPTH-(53-84) also induced osteoclast-like cell formation, but the maximal effect was only 30% as great as that of rPTH-(1-34). Importantly, rPTH-(1-34) induced TRAP+ MNC formation even when PTH1R-/- osteoclast progenitors (from fetal liver of mice homozygous for ablation of the PTH1R gene) were cocultured with MS1 cells. We conclude that activation of PTH1Rs on cells of the osteoclast lineage is not required for PTH-(1-34)-induced osteoclast formation in the presence of appropriate PTH-responsive marrow stromal cells. MS1 cells provide a useful model for further study of PTH regulation of osteoclastogenesis.     

Renal expression of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP receptor in a rat model of tubulointerstitial damage

The effect of a meniscal bearing on knee laxity in anterior cruciate ligament-sacrificing total knee arthroplasty was evaluated in 7 cadaver knees using a knee testing device that measured knee flexion angle as well as laxity to medial-lateral, anterior-posterior [AP], and rotational loads. A standard fixed tibial component and mobile tibial components (AP sliding, rotationally sliding, and AP and rotationally sliding) were used to evaluate AP, rotational, and varus-valgus stability and maximal flexion and extension with the neutrally positioned and malrotated tibial tray. The AP movable components increased AP laxity, and the fixed component decreased rotational laxity significantly when compared with the normal knees. The rotationally movable components did not change knee laxities significantly even when the tibial tray was malrotated. No significant difference among the components was detected when the maximal flexion and extension angles were compared in the neutrally positioned tibial tray. Malrotation of the tibial tray decreased the maximal extension angle in the fixed component. This study showed that the rotationally movable component can achieve near-normal laxity regardless of tibial tray rotation, but AP mobility of the bearing produces AP laxity that could lead to implant failure.     

Parathyroid hormone-related protein (PTHrP) action in rat articular chondrocytes: comparison of PTH(1-34), PTHrP(1-34), PTHrP(1-141), PTHrP(100-114) and antisense oligonucleotides against PTHrP

Parathyroid hormone-related protein (PTHrP) is thought to be an important autocrine/paracrine factor for chondrocyte metabolism since mice lacking the PTHrP gene exhibit abnormal cartilage development. To determine the biological role of PTHrP in chondrocytes, we first compared the agonist potency of human (h) PTHrP(1-34) with hPTH(1-34) in cultured rat articular chondrocytes. Neither hPTHrP(1-34) nor hPTH(1-34) altered basal DNA synthesis, but attenuated the stimulatory effect of transforming growth factor beta (TGF-beta). Both agents suppressed the expression of alpha(1) type II collagen mRNA in a dose-response fashion with the same potency. In addition, the action of exogenously added hPTHrP(1-34) and hPTH(1-34) on intracellular cAMP and [Ca2+]i levels was similar. We next compared the effect of PTHrP within its entire amino acid sequence (1-141). With regard to thymidine incorporation, alpha(1) type II collagen gene expression and accumulation of cAMP and [Ca2+]i level, there was no significant difference between hPTHrP(1-34) and hPTHrP(1-141). PTHrP C-terminal (100-114) did not show any function. To further investigate PTHrP function, intracellular PTHrP translation was inhibited by a transgene of antisense oligonucleotides against PTHrP. Antisense oligonucleotides decreased PTHrP mRNA translation, specifically inhibited DNA synthesis in control as well as TGF-beta-treated chondrocytes and enhanced alpha(1) type II collagen mRNA expression in TGF-beta-treated chondrocytes. These results suggest that there is no significant difference between exogenously added hPTH(1-34), hPTHrP(1-34) and PTHrP(1-141) with regard to the biological action of these agents, including cell growth, differentiation and second messenger pathway. However, the result of DNA synthesis in the antisense PTHrP-inhibition study suggests that intracellular PTHrP may have an as yet unknown biological role, in addition to a classical PTH/PTHrP receptor-mediated function in the rat articular chondrocyte.     

Analysis of liver regeneration in mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not type 2 receptor

We used KO mice lacking either TNF receptor 1 (TNFR-1) or receptor 2 (TNFR-2) to determine whether signaling at the start of liver regeneration after partial hepatectomy (PH) involves only one or both TNF receptors and to analyze in more detail the abnormalities caused by lack of TNFR-1 receptor, which is required for the initiation of liver regeneration. Lack of TNFR-2 had little effect on NF-kappaB and STAT3 binding, and no effect in interleukin-6 production after PH, but caused a delay in AP-1 and C/EBP binding and in the expression of c-jun and c-myc messenger RNA (mRNA). In contrast to mice lacking TNFR-1, which had deficient hepatocyte DNA synthesis and massive lipid accumulation in hepatocytes, TNFR-2 KO mice had normal liver structure and similar levels of hepatocyte DNA replication as those of wild type mice. We conclude that TNFR-1, but not TNFR-2, is necessary for liver regeneration, and that NF-kappaB and STAT3 binding are activated by signals transduced by TNFR-1. Inhibition of AP-1 and C/EBP binding and in the expression of c-jun and c-myc mRNA in the first 4 hours after PH, as well as the apparent lack of Fos in AP-1 complexes, had no effect on the timing or extent of DNA replication.     

Mice lacking the type I interleukin-1 receptor do not lose bone mass after ovariectomy

We measured the effects of ovariectomy on the bone mass of mice that lacked type I interleukin-1 receptor (IL-I R1 -/- mice) in two genetic backgrounds (C57BL/6 x 129/Sv and C57BL/6) to investigate the role of interleukin-1 in the actions of estrogen on bone. At three weeks after surgery, ovariectomized wild-type mice decreased trabecular bone volume in the proximal humerus by 70% in a C57BL/6 x 129/Sv background and 48% in a C57BL/6 background compared to sham-operated controls. In contrast, there was no significant decrease in trabecular bone mass in IL-1 R1 -/- mice after ovariectomy. The estrogen status of all groups was confirmed by measurement of uterine wet weight. These results demonstrate that a functional IL-1 response pathway is required for mice to lose trabecular bone mass after ovariectomy in this model and they imply that IL-1 is an important mediator of the effects of ovariectomy on bone mass. Hence, therapeutic interventions that block the effects of IL-1 on bone may be beneficial for treating diseases of rapid bone loss such as post-menopausal osteoporosis.     

Cell-specific signal transduction of parathyroid hormone (PTH)-related protein through stably expressed recombinant PTH/PTHrP receptors in vascular smooth muscle cells

PTH-related protein activates a G protein-coupled PTH/PTHrP receptor in many cell types and produces diverse biological actions. To study the signal transduction events associated with biological activity of the PTH/PTHrP receptor in vascular smooth muscle, a principal PTHrP-responsive tissue, rat aortic smooth muscle cells (A10) were stably transfected with a plasmid encoding a PTH/PTHrP receptor and tested for ligand binding, PTHrP-(1-34)-induced cAMP levels, inositol phosphate production, and cytosolic calcium transients. Of nineteen G418-resistant lines recovered, all exhibited high affinity binding [approximately dissociation constant (Kd) > 10(-10)) of iodinated [Tyr36]hPTHrP(1-36)NH2 and ligand-induced cAMP accumulation (2- to 100-fold), which was directly proportional to PTH/PTHrP receptor number (range 4 x 10(3) to 7 x 10(7) sites/cell]. PTHrP had no effect on intracellular calcium or inositol phosphate formation in any cell line regardless of receptor number despite the presence of detectable G alpha q). Transient overexpression of individual G alpha q proteins (G alpha q, G alpha 11 or G alpha 14) into PTH/PTHrP receptor-expressing A10 cells conferred the ability of PTHrP to increase intracellular calcium and inositol phosphate formation. Ligand activation of the recombinant PTH/PTHrP receptor elicited appropriate downstream biological effects in A10 cells including inhibition of DNA synthesis and osteopontin messenger RNA (mRNA) expression. Thus, a single PTH/PTHrP receptor, though capable of coupling to different G proteins, signals exclusively through a cAMP-dependent pathway in vascular smooth muscle.     

Type-1 parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptors activate phospholipase C in response to carboxyl-truncated analogs of PTH(1-34)

The carboxyl(C)-truncated human (h) PTH (hPTH) analog hPTH(1-31), which activates adenylyl cyclase (AC), but not protein kinase C, in rat osteosarcoma cells, exerts an anabolic effect on rat bone in vivo similar to that of hPTH(1-34). It has been proposed, therefore, that this action of PTH(1-34) is mediated exclusively by stimulation of AC via the rat type-1 PTH/PTH-related peptide (PTHrP) receptor (PTH1R). To determine whether this selective signaling pattern also might be a property of the hPTH1R, we studied signal transduction via heterologously expressed hPTH1Rs in response to activation by hPTH(1-34), hPTH(1-31), and a C-truncated analog that does not increase rat bone mass in vivo, hPTH(1-30). In porcine LLC-PK1 cells that stably expressed recombinant hPTH1Rs, these three peptides activated AC identically (EC50 = 1-2 nM). In cells with comparable expression of rat PTH1Rs, AC activation by hPTH(1-34) and hPTH(1-31) again was identical, whereas full activation by hPTH(1-30) required higher concentrations (EC50 = 10 nM vs. 1 nM). Surprisingly, hPTH(1-31) fully stimulated phospholipase C (PLC), via both species of PTH1Rs, with potency that was similar (hPTH1Rs) or slightly reduced (rat PTH1Rs), relative to that of hPTH(1-34). hPTH(1-30), however, was 5-fold less potent than hPTH(1-34) in activating PLC via hPTH1Rs and showed weak and only partial activity via the rat PTH1R. Comparable results were obtained when human and rat PTH1Rs were transiently expressed heterologously in COS-7 cells or homologously in HEK 293 and UMR 106-01 cells, respectively. Binding affinities of these C-truncated peptides to human and rat PTH1Rs were concordant with their relative potencies in activating PLC. We conclude that hPTH(1-31) and, to a lesser extent, hPTH(1-30) can activate PLC, as well as AC, via both rat and human PTH1Rs. Accordingly, a role for PLC activation in the anabolic action of PTH in vivo cannot be excluded.     

Deficient liver regeneration after carbon tetrachloride injury in mice lacking type 1 but not type 2 tumor necrosis factor receptor

Signaling by tumor necrosis factor type 1 receptor (TNFR-1) is required for the initiation of liver regeneration after partial hepatectomy. Using knockout mice that lack either TNFR-1 or TNFR-2, we determined whether signaling through TNF receptors is important for liver injury and hepatocyte proliferation induced by carbon tetrachloride (CCl4). Lack of TNFR-1 inhibited hepatocyte DNA synthesis after CCl4 injection. At 44 hours after the injection, replication of hepatocytes in TNFR-1 was 50% to 90% lower than in wild-type (WT) animals, depending on the dose injected. In WT animals, hepatocyte replication was essentially completed by 4 days after CCl4 injection, but replication at a low level persisted in TNFR-1 mice for at least 2 weeks. TNFR-1 knockout mice had little detectable NF-kappa B and STAT3 binding during the first 5 hours after CCl4, high plasma TNF, and reduced levels of plasma interleukin (IL)-6 and liver IL-6 mRNA. Injection of IL-6 30 minutes before CCl4 administration corrected the deficiency of hepatocyte replication at 44 hours and restored STAT3 binding to normal levels. In contrast, mice lacking TNFR-2 did not differ significantly from WT mice in NF-kappa B and STAT3 binding, IL-6 and TNF levels, or hepatocyte replication. Although AP-1 binding was induced in WT TNFR-1 and TNFR-2 knockout mice, binding in TNFR-2 knockouts was lower than in WT mice. C/EBP binding was much lower in TNFR-1 and TNFR-2 knockout mice than in WT mice. As assessed by morphological analysis and alanine aminotransferase levels, the acute injury caused by CCl4 appeared to be similar in the three groups of animals, but subsequent regeneration was impaired in mice lacking TNFR-1. We conclude that a TNFR-1 signaling pathway involving NF-kappa B, IL-6, and STAT3 is an important component of the hepatocyte mitogenic response induced by CCl4 injury in mouse liver.     

The parathyroid hormone (PTH)/PTH-related peptide receptor mediates actions of both ligands in murine bone

PTH and PTH-related peptide (PTHrP) have been shown to bind to and activate the same PTH/PTHrP receptor. Recent studies have demonstrated, however, the presence of additional receptors specific for each ligand. We used the PTHrP and PTH/PTHrP receptor gene knock-out models to investigate whether this receptor mediates the actions of both ligands in bone. The similar phenotype of the PTHrP (-/-) and PTH/PTHrP receptor (-/-) animals in the growth plate of the tibia suggests that this receptor mediates the actions of PTHrP. Electron microscopic studies have confirmed the accelerated differentiation and disordered organization of chondrocytes, with the accumulation of large amounts of dispersed glycogen granules in the cytoplasm of proliferative and maturing cells of both genotypes. The contrasting growth plate mineralization patterns of the PTHrP (-/-) and PTH/PTHrP receptor (-/-) mice, however, suggest that the actions of PTHrP and the PTH/PTHrP receptor are not identical. Studies using calvariae from PTH/PTHrP receptor (-/-) embryos demonstrate that this receptor solely mediates the ability of PTH and PTHrP to stimulate adenylate cyclase in bone and to stimulate bone resorption. Furthermore, we show that osteoblasts of PTH/PTHrP receptor (-/-) animals, but not PTHrP (-/-) animals, have decreased levels of collagenase 3, osteopontin, and osteocalcin messenger RNAs. The PTH/PTHrP receptor, therefore, mediates distinct physiologic actions of both PTH and PTHrP.     

Bone morphogenetic proteins and bFGF exert opposing regulatory effects on PTHrP expression and inorganic pyrophosphate elaboration in immortalized murine endochondral hypertrophic chondrocytes (MCT cells)

We isolated cDNA clones encoding five S-RNases (S1-, S3-, S5-, S6-, S7-RNases) from pistils of Pyrus pyrifolia (Japanese pear), a member of the Rosaceae. Their amino acid sequences were aligned with those of other rosaceous S-RNases sequenced so far. A total of 76 conserved amino acid residues were stretched throughout the sequence, but were absent from the 51-66 region which was designated the hypervariable (HV) region. The phylogenetic tree of rosaceous S-RNases showed that S-RNase polymorphism predated the divergence of Pyrus and Malus. Pairwise comparison of these S-RNases detected two highly homologous pairs, P. pyrifolia S1- and S4-RNases (90.0%) and P. pyrifolia S3- and S5-RNases (95.5%). The positions of amino acid substitutions between S1- and S4-RNases were spread over the entire region, but in the pair of S3- and S5-RNases, amino acid substitutions were found in the 21-90 region including the HV region. The substitutions in this restricted region appear to be sufficient to discriminate between S3 and S5 pollen and to trigger the self-incompatible reaction.     

Conditionally immortalized neural progenitor cells grafted to the striatum exhibit site-specific neuronal differentiation and establish connections with the host globus pallidus

The cell line RN33B has been reported to differentiate into neurons in a site-specific manner when grafted to the cortex and hippocampus of adult rats. To investigate the fate of RN33B cells in a subcortical structure, we grafted RN33B cells into the intact or excitoxically lesioned striatum of adult or neonatal rats. The total number and phenotypic characteristics of the [3H]thymidine-labeled grafted cells were analyzed at different time points after transplantation. Transplanted RN33B cells were found to survive, integrate, and differentiate into both neurons and astrocytes, and a significant proportion of the cells (approx. 10%) were found to differentiate into cells with morphological and phenotypic characteristics of medium-sized striatal projection neurons. Retrograde tracing showed that at least some of the graft-derived neurons were capable of establishing connections with one of the primary striatal targets, the globus pallidus. These findings demonstrate a remarkable capacity of the RN33B cells for site-specific neuronal differentiation in both the adult and the developing striatum and suggest that the same differentiating factors that are operating during normal neurogenesis in brain development are retained, at least to some extent, also in the adult CNS.     

Inhibitory activity of IL-1 receptor antagonist depends on the balance between binding capacity for IL-1 receptor type 1 and IL-1 receptor type II

A series of mutants of human IL-1 receptor antagonist (IL-1ra) has been designed by comparison of IL-1ra and IL-1beta structures in order to increase receptor antagonist capacity. Upon in vitro and in vivo assay of IL-1 antagonism, the IL-1ra mutants DoB 0039 (N91-->R), DoB 0040 (T109-->A) and DoB 0041 (N91/T109-->R/A) could inhibit IL-1beta effects more efficiently than wild-type IL-1ra, with DoB 0041 being the most active. Analysis of the receptor-binding capacity of the IL-1ra mutants showed that all three mutants could inhibit binding of IL-1alpha or IL-1beta to IL-1RI-bearing cells more efficiently than wild-type IL-1ra. Conversely, binding of IL-1beta to IL-1RII-bearing cells could be inhibited by DoB 0041 much less efficiently than by wild-type IL-1ra. It is known that the two types of IL-1 receptors (IL-1RI and IL-1RII) play different roles in the regulation of IL-1 activity, with IL-1RI being solely responsible for cell triggering upon IL-1 binding, whereas IL-1RII acts as a scavenger of IL-1 and can thus be considered as a natural IL-1 inhibitor. Thus, the enhanced inhibitory capacity of DoB 0041 as compared with wild-type IL-1ra is explained in terms of better binding to the activating receptor IL-1RI and poorer interaction with the inhibitory receptor IL-1RII.     

Fas/Apo-1 (CD95) receptor lacking the intracytoplasmic signaling domain protects tumor cells from Fas-mediated apoptosis

Despite improved preservation methods, graft dysfunction after liver transplantation continues to contribute considerably to postoperative morbidity and mortality. In clinical and experimental studies prostaglandin (PG)I2 analogs proved effective in the treatment of liver damage of different origin. Using in vivo fluorescence microscopy in a rat liver transplantation model, we studied the effect of donor bolus pretreatment with the PGI2 analog epoprostenol on hepatic graft revascularization. After epoprostenol bolus pretreatment (group 1: liver transplantation/PGI2), perfusion of liver sinusoids after reperfusion was significantly improved as compared with untreated donor livers (group 2: liver transplantation (95.2+/-0.6% vs. 75.3+/-3.8%, mean +/- SEM; P=0.001) and epoprostenol was found almost in the range of that in normal nontransplanted livers (99.4+/-0.2%). In addition, leukocyte adherence in liver lobules (21.0+/-3.5 vs. 115+/-11.5 n/lobule; P=0.001) and postsinusoidal venules (23.0+/-3.8 vs. 113+/-11.3 n/mm2 endothelial surface; P=0.002) was significantly reduced in the pretreated grafts. Bile production in the recipient was significantly increased by epoprostenol pretreatment of the donor (1.88+/-0.4 vs. 0.63+/-0.13 g/100 g liver*1 hr; P=0.015), indicating restored liver function. These results suggest that the prostacyclin analog epoprostenol is effective in preconditioning the graft prior to transplantation, i.e., improving preservation and increasing graft resistance to ischemia/reperfusion injury. Thus, favorable effects on early graft function after clinical liver transplantation may be achieved by introducing epoprostenol pretreatment into the harvesting procedure.