Zinc Supplementation Increases Procollagen Type 1 Amino-Terminal Propeptide in Premenarcheal Girls: A Randomized Controlled Trial.

BACKGROUND: Data have shown that healthy children and adolescents have an inadequate intake of zinc, an essential nutrient for growth. It is unclear whether zinc supplementation can enhance bone health during this rapid period of growth and development. OBJECTIVE: The primary aim of this study was to determine the effect of zinc supplementation on biochemical markers of bone turnover and growth in girls entering the early stages of puberty. The secondary aim was to test moderation by race, body mass index (BMI) classification, and plasma zinc status at baseline. METHODS: One hundred forty seven girls aged 9-11 y (46% black) were randomly assigned to a daily oral zinc tablet (9 mg elemental zinc; n = 75) or an identical placebo (n = 72) for 4 wk. Fasting plasma zinc, procollagen type 1 amino-terminal propeptide (P1NP; a bone formation marker), carboxy-terminal telopeptide region of type 1 collagen (ICTP; a bone resorption marker), and insulin-like growth factor I (IGF-I) were assessed at baseline and post-test. Additional markers of bone formation (osteocalcin) and resorption (urinary pyridinoline and deoxypyridinoline) were also measured. RESULTS: Four weeks of zinc supplementation increased plasma zinc concentrations compared with placebo [mean change, 1.8 µmol/L (95% CI: 1.0, 2.6) compared with 0.2 µmol/L (95% CI: -0.3, 0.7); P < 0.01]. Zinc supplementation also increased serum P1NP concentrations compared with placebo [mean change, 23.8 µmol/L (95% CI: -14.9, 62.5) compared with -31.0 µmol/L (95% CI: -66.4, 4.2); P = 0.04). There was no effect from zinc supplementation on osteocalcin, ICTP, pyridinoline, deoxypyridinoline, or IGF-I. There was no moderation by race, BMI classification, or plasma zinc status at baseline. CONCLUSIONS: Our data suggest that 4 wk of zinc supplementation increases bone formation in premenarcheal girls. Further studies are needed to determine whether supplemental zinc can improve childhood bone strength. This trial was registered at clinicaltrials.gov as NCT01892098.

Impact of dietary aromatic amino acids on osteoclastic activity.

We had shown that aromatic amino acid (phenylalanine, tyrosine, and tryptophan) supplementation prevented bone loss in an aging C57BL/6 mice model. In vivo results from the markers of bone breakdown suggested an inhibition of osteoclastic activity or differentiation. To assess osteoclastic differentiation, we examined the effects of aromatic amino acids on early /structural markers as vitronectin receptor, calcitonin receptor, and carbonic anhydrase II as well as, late/functional differentiation markers; cathepsin K and matrix metalloproteinase 9 (MMP-9). Our data demonstrate that the aromatic amino acids down-regulated early and late osteoclastic differentiation markers as measured by real time PCR. Our data also suggest a link between the vitronectin receptor and the secreted cathepsin K that both showed consistent effects to the aromatic amino acid treatment. However, the non-attachment related proteins, calcitonin receptor, and carbonic anhydrase II, demonstrated less consistent effects in response to treatment. Our data are consistent with aromatic amino acids down-regulating osteoclastic differentiation by suppressing remodeling gene expression thus contributing initially to the net increase in bone mass seen in vivo.

Age-related increase of kynurenine enhances miR29b-1-5p to decrease both CXCL12 signaling and the epigenetic enzyme Hdac3 in bone marrow stromal cells.

Mechanisms leading to age-related reductions in bone formation and subsequent osteoporosis are still incompletely understood. We recently demonstrated that kynurenine (KYN), a tryptophan metabolite, accumulates in serum of aged mice and induces bone loss. Here, we report on novel mechanisms underlying KYN's detrimental effect on bone aging. We show that KYN is increased with aging in murine bone marrow mesenchymal stem cells (BMSCs). KYN reduces bone formation via modulating levels of CXCL12 and its receptors as well as histone deacetylase 3 (Hdac3). BMSCs responded to KYN by significantly decreasing mRNA expression levels of CXCL12 and its cognate receptors, CXCR4 and ACKR3, as well as downregulating osteogenic gene RUNX2 expression, resulting in a significant inhibition in BMSCs osteogenic differentiation. KYN's effects on these targets occur by increasing regulatory miRNAs that target osteogenesis, specifically miR29b-1-5p. Thus, KYN significantly upregulated the anti-osteogenic miRNA miR29b-1-5p in BMSCs, mimicking the up-regulation of miR-29b-1-5p in human and murine BMSCs with age. Direct inhibition of miR29b-1-5p by antagomirs rescued CXCL12 protein levels downregulated by KYN, while a miR29b-1-5p mimic further decreased CXCL12 levels. KYN also significantly downregulated mRNA levels of Hdac3, a target of miR-29b-1-5p, as well as its cofactor NCoR1. KYN is a ligand for the aryl hydrocarbon receptor (AhR). We hypothesized that AhR mediates KYN's effects in BMSCs. Indeed, AhR inhibitors (CH-223191 and 3',4'-dimethoxyflavone [DMF]) partially rescued secreted CXCL12 protein levels in BMSCs treated with KYN. Importantly, we found that treatment with CXCL12, or transfection with an miR29b-1-5p antagomir, downregulated the AhR mRNA level, while transfection with miR29b-1-5p mimic significantly upregulated its level. Further, CXCL12 treatment downregulated IDO, an enzyme responsible for generating KYN. Our findings reveal novel molecular pathways involved in KYN's age-associated effects in the bone microenvironment that may be useful translational targets for treating osteoporosis.

Monomethylfumarate protects against ovariectomy-related changes in body composition.

Osteoporosis, low bone mass that increases fracture susceptibility, affects approximately 75 million individuals in the United States, Europe and Japan, with the number of osteoporotic fractures expected to increase by more than 3-fold over the next 50 years. Bone mass declines with age, although the mechanisms for this decrease are unclear. Aging enhances production of reactive oxygen species, which can affect bone formation and breakdown. The multiple sclerosis drug Tecfidera contains dimethylfumarate, which is rapidly metabolized to monomethylfumarate (MMF); MMF is thought to function through nuclear factor erythroid-derived-2-like-2 (Nrf2), a transcription factor activated by oxidative stress which induces the expression of endogenous anti-oxidant systems. We hypothesized that MMF-elicited increases in anti-oxidants would inhibit osteopenia induced by ovariectomy, as a model of aging-related osteoporosis and high oxidative stress. We demonstrated that MMF activated Nrf2 and induced anti-oxidant Nrf2 target gene expression in bone marrow-derived mesenchymal stem cells. Sham-operated or ovariectomized adult female mice were fed chow with or without MMF and various parameters monitored. Ovariectomy produced the expected effects, decreasing bone mineral density and increasing body weight, fat mass, bone marrow adiposity and serum receptor activator of nuclear factor-kappa-B ligand (RANKL) levels. MMF decreased fat but not lean mass. MMF improved trabecular bone microarchitecture after adjustment for body weight, although the unadjusted data showed few differences; MMF also tended to increase adjusted cortical bone and to reduce bone marrow adiposity and serum RANKL levels. Because these results suggest the possibility that MMF might be beneficial for bone, further investigation seems warranted.

The glucocorticoid receptor in osteoprogenitors regulates bone mass and marrow fat.

Excess fat within bone marrow is associated with lower bone density. Metabolic stressors such as chronic caloric restriction (CR) can exacerbate marrow adiposity, and increased glucocorticoid signaling and adrenergic signaling are implicated in this phenotype. The current study tested the role of glucocorticoid signaling in CR-induced stress by conditionally deleting the glucocorticoid receptor (GR) in bone marrow osteoprogenitors (Osx1-Cre) of mice subjected to CR and ad libitum diets. Conditional knockout of the GR (GR-CKO) reduced cortical and trabecular bone mass as compared to wildtype (WT) mice under both ad libitum and CR conditions. No interaction was detected between genotype and diet, suggesting that the GR is not required for CR-induced skeletal changes. The lower bone mass in GR-CKO mice, and the further suppression of bone by CR, resulted from suppressed bone formation. Interestingly, treatment with the -adrenergic receptor antagonist propranolol mildly but selectively improved metrics of cortical bone mass in GR-CKO mice during CR, suggesting interaction between adrenergic and glucocorticoid signaling pathways that affects cortical bone. GR-CKO mice dramatically increased marrow fat under both ad libitum and CR-fed conditions, and surprisingly propranolol treatment was unable to rescue CR-induced marrow fat in either WT or GR-CKO mice. Additionally, serum corticosterone levels were selectively elevated in GR-CKO mice with CR, suggesting the possibility of bone-hypothalamus-pituitary-adrenal crosstalk during metabolic stress. This work highlights the complexities of glucocorticoid and ß-adrenergic signaling in stress-induced changes in bone mass, and the importance of GR function in suppressing marrow adipogenesis while maintaining healthy bone mass.

Deletion of protein kinase D1 in osteoprogenitor cells results in decreased osteogenesis in vitro and reduced bone mineral density in vivo.

Protein kinase D1 (PRKD1) is thought to play a role in a number of cellular functions, including proliferation and differentiation. We hypothesized that PRKD1 in bone marrow-derived mesenchymal stem cells (BMMSC) could modulate osteogenesis. In BMMSCs from floxed PRKD1 mice, PRKD1 ablation with adenovirus-mediated Cre-recombinase expression inhibited BMMSC differentiation in vitro. In 3- and 6-month-old conditional knockout mice (cKO), in which PRKD1 was ablated in osteoprogenitor cells by osterix promoter-driven Cre-recombinase, bone mineral density (BMD) was significantly reduced compared with floxed control littermates. Microcomputed tomography analysis also demonstrated a decrease in trabecular thickness and bone volume fraction in cKO mice at these ages. Dynamic bone histomorphometry suggested a mineralization defect in the cKO mice. However, by 9 months of age, the bone appeared to compensate for the lack of PRKD1, and BMD was not different. Taken together, these results suggest a potentially important role for PRKD1 in bone formation.

Protein kinase D1 conditional null mice show minimal bone loss following ovariectomy.

We previously found that 3- and 6-month-old male mice with conditional ablation of protein kinase D1 (PRKD1) in osteoprogenitor cells (expressing Osterix) exhibited reduced bone mass. Others have demonstrated similar effects in young female PRKD1-deficient mice. Here we examined the bone resorptive response of adult female floxed control and conditional knockout (cKO) mice undergoing sham surgery or ovariectomy (OVX). Femoral and tibial bone mineral density (BMD) values were significantly reduced upon OVX in control, but not cKO, females compared to the respective sham-operated mice. Micro-CT analysis showed that OVX significantly increased trabecular number and decreased trabecular spacing in cKO but not control mice. Finally, in control mice serum levels of a marker of bone resorption (pyridinoline crosslinks) and the osteoclast activator RANKL significantly increased upon OVX; however, no such OVX-induced increase was observed in cKO mice. Our results suggest the potential importance of PRKD1 in response to estrogen loss in bone.

Amino acids as signaling molecules modulating bone turnover.

Except for the essential amino acids (AAs), much of the focus on adequate dietary protein intake has been on total nitrogen and caloric intake rather than AA composition. Recent data, however, demonstrate that "amino-acid sensing" can occur through either intracellular or extracellular nutrient-sensing mechanisms. In particular, members of the class 3 G-protein coupled receptor family, like the calcium-sensing receptor are known to preferentially bind specific AAs, which then modulate receptor activation by calcium ions and thus potentially impact bone turnover. In pursuing the possibility of direct nutrient effects on bone cells, we examined individual AA effects on osteoprogenitor/bone marrow stromal cells (BMSCs), a key target for bone anabolism. We demonstrate that BMSCs express both intracellular and extracellular nutrient sensing pathways and that AAs are required for BMSC survival. In addition, certain AA types, like members of the aromatic AAs, can potently stimulate increases in intracellular calcium and ERK phosphorylation/activation. Further, based on the in vitro data, we examined the effect of specific AAs on bone mass. To better evaluate the impact of specific AAs, we added these to a low-protein diet. Our data demonstrate that a low-protein diet itself is associated with a significant drop in bone mineral density (BMD) in the older mice, related, at least in part, to an increase in osteoclastic activity. This drop in BMD in mice on the low-protein diet is prevented by addition of AAs from the aromatic group. Taken together our data show that AAs function as specific and selective signaling molecules in bone cells.

Glucose-dependent insulinotropic peptide-overexpressing transgenic mice have increased bone mass.

Glucose-dependent insulinotropic peptide (GIP) is an intestinally secreted hormone the release of which is stimulated by nutrient ingestion. We previously reported that GIP receptors are present in osteoblastic cells and that GIP increases collagen type I synthesis and alkaline phosphatase activity in isolated osteoblasts. We have also shown that osteoclasts express GIP receptors and that GIP inhibits osteoclastic activity and differentiation. In addition, using GIP receptor knockout mice we demonstrated that absence of GIP receptor signaling resulted in a low bone mass phenotype. To further define GIP's role as an anabolic hormone in vivo, we utilized a genetically altered mouse model, a transgenic mouse overexpressing GIP under the control of the metallothionein promoter (Tg+). Tg+ mice had significantly higher mean GIP levels even in the absence of added dietary zinc. Tg+ animals also had a significant increase in markers of bone formation and a decrease in markers of bone resorption. Consistent with these biochemical data, GIP transgenic mice had a significant increase in bone mass as measured by densitometry and histomorphometry. These data support the conclusion that GIP inhibits bone resorption and stimulates bone formation and that excess signaling through the GIP receptor results in gain of bone mass. In view of GIP's role in nutrient absorption, our data suggest that this hormone may serve an important role in linking nutrient ingestion to bone formation.

Kynurenine, a Tryptophan Metabolite That Accumulates With Age, Induces Bone Loss.

Age-dependent bone loss occurs in humans and in several animal species, including rodents. The underlying causal mechanisms are probably multifactorial, although an age-associated increase in the generation of reactive oxygen species has been frequently implicated. We previously reported that aromatic amino acids function as antioxidants, are anabolic for bone, and that they may potentially play a protective role in an aging environment. We hypothesized that upon oxidation the aromatic amino acids would not only lose their anabolic effects but also potentially become a catabolic byproduct. When measured in vivo in C57BL/6 mice, the tryptophan oxidation product and kynurenine precursor, N-formylkynurenine (NFK), was found to increase with age. We tested the direct effects of feeding kynurenine (kyn) on bone mass and also tested the short-term effects of intraperitoneal kyn injection on bone turnover in CD-1 mice. µCT analyses showed kyn-induced bone loss. Levels of serum markers of osteoclastic activity (pyridinoline [PYD] and RANKL) increased significantly with kyn treatment. In addition, histological and histomorphometric studies showed an increase in osteoclastic activity in the kyn-treated groups in both dietary and injection-based studies. Further, kyn treatment significantly increased bone marrow adiposity, and BMSCs isolated from the kyn-injected mice exhibited decreased mRNA expression of Hdac3 and its cofactor NCoR1 and increased expression of lipid storage genes Cidec and Plin1. A similar pattern of gene expression is observed with aging. In summary, our data show that increasing kyn levels results in accelerated skeletal aging by impairing osteoblastic differentiation and increasing osteoclastic resorption. These data would suggest that kyn could play a role in age-induced bone loss. © 2017 American Society for Bone and Mineral Research.

Insulin Resistance and the IGF-I-Cortical Bone Relationship in Children Ages 9 to 13 Years.

IGF-I is a pivotal hormone in pediatric musculoskeletal development. Although recent data suggest that the role of IGF-I in total body lean mass and total body bone mass accrual may be compromised in children with insulin resistance, cortical bone geometric outcomes have not been studied in this context. Therefore, we explored the influence of insulin resistance on the relationship between IGF-I and cortical bone in children. A secondary aim was to examine the influence of insulin resistance on the lean mass-dependent relationship between IGF-I and cortical bone. Children were otherwise healthy, early adolescent black and white boys and girls (ages 9 to 13 years) and were classified as having high (n = 147) or normal (n = 168) insulin resistance based on the homeostasis model assessment of insulin resistance (HOMA-IR). Cortical bone at the tibia diaphysis (66% site) and total body fat-free soft tissue mass (FFST) were measured by peripheral quantitative computed tomography (pQCT) and dual-energy X-ray absorptiometry (DXA), respectively. IGF-I, insulin, and glucose were measured in fasting sera and HOMA-IR was calculated. Children with high HOMA-IR had greater unadjusted IGF-I (p < 0.001). HOMA-IR was a negative predictor of cortical bone mineral content, cortical bone area (Ct.Ar), and polar strength strain index (pSSI; all p ≤ 0.01) after adjusting for race, sex, age, maturation, fat mass, and FFST. IGF-I was a positive predictor of most musculoskeletal endpoints (all p < 0.05) after adjusting for race, sex, age, and maturation. However, these relationships were moderated by HOMA-IR (pInteraction < 0.05). FFST positively correlated with most cortical bone outcomes (all p < 0.05). Path analyses demonstrated a positive relationship between IGF-I and Ct.Ar via FFST in the total cohort (ßIndirect Effect = 0.321, p < 0.001). However, this relationship was moderated in the children with high (ßIndirect Effect = 0.200, p < 0.001) versus normal (ßIndirect Effect = 0.408, p < 0.001) HOMA-IR. These data implicate insulin resistance as a potential suppressor of IGF-I-dependent cortical bone development, though prospective studies are needed. © 2017 American Society for Bone and Mineral Research.

Age-related loss of muscle mass and bone strength in mice is associated with a decline in physical activity and serum leptin.

The mechanisms underlying age-related loss of muscle and bone tissue are poorly understood but are thought to involve changes in sex hormone status, physical activity, and circulating levels of inflammatory cytokines. This study attempts to develop an animal model useful for evaluating these mechanisms in vivo. Male C57BL/6 mice were included for study at 3, 6, 12, 18, 24, and 29 months of age. Endocortical mineralizing surface, serum leptin, body weight, and percentage of body fat all increased between 6 and 12 months of age as activity level declined. Serum levels of the inflammatory marker IL-6 increased significantly after 12 months of age, following the observed increase in body weight and percent body fat. Hindlimb muscle mass declined significantly between 18 and 24 months of age, both absolutely and relative to total body mass, with a further decline ( approximately 15%) between 24 and 29 months. Loss of muscle mass after 18 months of age was accompanied by a significant increase in bone resorption, as indicated by serum pyridinoline cross-links, and a significant decrease in fat mass, serum leptin, bone strength, bone mineral density, and vertical cage activity. No significant changes in serum testosterone with aging were detected in the mice, as levels were essentially constant between 6 and 29 months. Our data show that mice lose a significant amount of muscle and bone tissue with age, and this loss of musculoskeletal tissue is accompanied by a drop in serum leptin and preceded by a significant decrease in physical activity.

Effects of glucose-dependent insulinotropic peptide on behavior.

Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone that rises rapidly in response to nutrient ingestion. The GIP receptor is widely expressed in the brain including the brain stem, telencephalon, diencephalon, olfactory bulb, pituitary, and cerebellum. Until recently it was not clear what the endogenous ligand for this receptor was because no GIP expression had been demonstrated in the brain. GIP synthesis has now been documented in the dentate gyrus of the hippocampus. To define GIP effects on behavior we utilized a mouse model a GIP-overexpressing transgenic mouse (GIP Tg). Specifically, anxiety-related behavior, exploration, memory, and nociception were examined. Compared to age-matched adult male C57BI/6 controls GIP Tg mice displayed enhanced exploratory behavior in the open-field locomotor activity test. GIP Tg mice also demonstrated increased performance in some of the motor function tests. These data suggest that the GIP receptor plays a role in the regulation of locomotor activity and exploration. To our knowledge, this is the first report of effects of GIP on behavior.

Multiple melanocortin receptors are expressed in bone cells.

Melanocortin receptors belong to the seven transmembrane domain, G-protein coupled family of receptors. There are five members of this receptor family labeled MC1R-MC5R. These receptors are activated by fragments derived from a larger molecule, proopiomelanocortin (POMC) and include ACTH, alpha beta and gamma-MSH and beta-endorphin. Because of in vitro and in vivo data suggesting direct effects of these POMC molecules on bone and bone turnover, we examined bone and bone derived cells for the presence of the various members of the melanocortin receptor family. We report that the five known melanocortin receptors are expressed to varying degrees in osteoblast-like and osteoclastic cells. POMC fragments increased proliferation and expression of a variety of genes in osteoblastic cells. Furthermore, POMC mRNA was detected in osteoclastic cells. These data demonstrate that POMC-derived peptide hormones acting through high affinity melanocortin receptors have specific effects on bone cells. Thus, in addition to the indirect effects of POMC-derived hormones on bone turnover through their modulation of steroid hormone secretion, POMC fragments may have direct and specific effects on bone cell subpopulations.

Glucose-dependent insulinotropic polypeptide receptor knockout mice have altered bone turnover.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone, which is secreted from endocrine cells in the small intestine after meal ingestion. GIP has been shown to affect osteoblastic function in vitro; however, the in vivo effects of GIP on bone remodeling remain unclear. In the present study, we investigated the role of GIP in modulating bone turnover, by evaluating serum markers of bone turnover, bone density, bone morphology, and changes in biomechanical bone strength over time (one to five months) in GIP receptor knockout mice (GIPR-/- mice). The GIPR-/- mice showed a decreased bone size, lower bone mass, altered bone microarchitecture and biomechanical properties, and altered parameters for bone turnover, especially in bone formation. Moreover, the effects of GIP on bone mass were site-specific and compensatory mechanism developed over time and ameliorated the impact of the loss of GIP signaling on bone mass. Further, GIPR-/- mice had earlier age-related changes than wild-type mice in body composition, including bone mass, lean body mass, and fat percentage. In summary, our results indicate that GIP has an anabolic effect on bone mass and bone quality and suggests that GIP may be a hormonal link between nutrient ingestion and utilization.

Carboxy-terminal PTH fragments stimulate [3H]thymidine incorporation in vascular endothelial cells.

We have previously reported that the intact PTH molecule (1-84) stimulates proliferation of human umbilical vein endothelial cells (HUVECs). To define the bioactive portion of the PTH molecule we utilized amino, mid and carboxy-terminal PTH fragments. Carboxy- but not amino-terminal fragments were equivalent to the intact PTH molecule in stimulating [3H]thymidine incorporation in HUVEC. Carboxy- but not amino-terminal PTH fragments increased intracellular calcium. Blocking the rise in intracellular calcium with calcium chelators abolished PTHs proliferative effect on HUVEC. In contrast to PTH 1-84, the carboxy-terminal fragment effect on [3H]thymidine incorporation was blocked by KN-93 an inhibitor of CaM kinase II. Taken together, these data suggest that the carboxy-terminal PTH is (or contains) the bioactive fragment responsible for the changes in intracellular calcium and thymidine incorporation in HUVEC stimulated with the intact PTH molecule.

Oxidation of the aromatic amino acids tryptophan and tyrosine disrupts their anabolic effects on bone marrow mesenchymal stem cells.

Age-induced bone loss is associated with greater bone resorption and decreased bone formation resulting in osteoporosis and osteoporosis-related fractures. The etiology of this age-induced bone loss is not clear but has been associated with increased generation of reactive oxygen species (ROS) from leaky mitochondria. ROS are known to oxidize/damage the surrounding proteins/amino acids/enzymes and thus impair their normal function. Among the amino acids, the aromatic amino acids are particularly prone to modification by oxidation. Since impaired osteoblastic differentiation from bone marrow mesenchymal stem cells (BMMSCs) plays a role in age-related bone loss, we wished to examine whether oxidized amino acids (in particular the aromatic amino acids) modulated BMMSC function. Using mouse BMMSCs, we examined the effects of the oxidized amino acids di-tyrosine and kynurenine on proliferation, differentiation and Mitogen-Activated Protein Kinase (MAPK) pathway. Our data demonstrate that amino acid oxides (in particular kynurenine) inhibited BMMSC proliferation, alkaline phosphatase expression and activity and the expression of osteogenic markers (Osteocalcin and Runx2). Taken together, our data are consistent with a potential pathogenic role for oxidized amino acids in age-induced bone loss.

Glucose-dependent insulinotropic peptide stimulates proliferation and TGF-beta release from MG-63 cells.

Glucose-dependent insulinotropic peptide (GIP) is known to modulate alkaline phosphatase activity and collagen type I message in osteoblastic-like cells. GIP effects on cell proliferation are not known. We report that GIP dose dependently stimulated 3H-thymidine incorporation in the osteoblastic-like cell line MG-63. Furthermore, GIP increased message and secretion of transforming growth factor beta (TGF-beta), an agent known to regulate osteoblastic proliferation and differentiation. However, when GIP was added to MG-63 cells concurrently with a TGF-beta neutralizing antibody, there was no effect on 3H-thymidine incorporation in these cells. These data demonstrate that GIP stimulates osteoblastic-like cell proliferation but that this effect is not mediated by TGF-beta.

Parathyroid hormone effects on signaling pathways in endothelial cells vary with peptide concentration.

We have previously reported that parathyroid hormone (PTH) has specific effects on a human umbilical vein endothelial cell line. Further studies were performed to characterize the signaling cascades initiated by PTH. We report that PTH induced the appearance of voltage sensitive calcium channels. Furthermore, PTH increased ceramide but not diacylglycerol content. Since elevations in [Ca(2+)](i) and phospholipid turnover are signals for the activation of protein kinase C (PKC), the cells were screened for PKC isoforms. PTH induced a redistribution of the PKCepsilon to the particulate fractions of cell homogenates. In summary, PTH induced PKC translocation through a calcium-phospholipid pathway in an endothelial cell line.

Targeted disruption of the Lasp-1 gene is linked to increases in histamine-stimulated gastric HCl secretion.

Lasp-1 (LIM and SH3 domain protein 1) is a multidomain actin-binding protein that is differentially expressed within epithelial tissues and brain. In the gastric mucosa, Lasp-1 is highly expressed in the HCl-secreting parietal cell, where it is prominently localized within the F-actin-rich subcellular regions. Histamine-induced elevation of parietal cell [cAMP]i increases Lasp-1 phosphorylation, which is correlated with activation of HCl secretion. To determine whether Lasp-1 is involved in the regulation of HCl secretion in vivo, we generated a murine model with a targeted disruption of the Lasp-1 gene. Lasp-1-null mice had slightly lower body weights but developed normally and had no overt phenotypic abnormalities. Basal HCl secretion was unaffected by loss of Lasp-1, but histamine stimulation induced a more robust acid secretory response in Lasp-1-null mice compared with wild-type littermates. A similar effect of histamine was observed in isolated gastric glands on the basis of measurements of accumulation of the weak base [14C]aminopyrine. In addition, inhibition of the acid secretory response to histamine by H2 receptor blockade with ranitidine proceeded more slowly in glands from Lasp-1-null mice. These findings support the conclusion that Lasp-1 is involved in the regulation of parietal HCl secretion. We speculate that cAMP-dependent phosphorylation of Lasp-1 alters interactions with F-actin and/or endocytic proteins that interact with Lasp-1, thereby regulating the trafficking/activation of the H+, K+-ATPase (proton pump).