Dose-dependent glycometabolic effects of sotagliflozin on type 1 diabetes over 12 weeks: The inTandem4 trial.

AIMS: To assess the dose-related effects of sotagliflozin, a novel dual inhibitor of sodium-glucose co-transporters-1 and -2, in type 1 diabetes (T1D). MATERIALS AND METHODS: In this 12-week, multicentre, randomized, double-blind, placebo-controlled dose-ranging trial, adults with T1D were randomized to once-daily placebo (n = 36) or sotagliflozin 75 mg (n = 35), 200 mg (n = 35) or 400 mg (n = 35). Insulin was maintained at baseline doses. The primary endpoint was least squares mean (LSM) change in glycated haemoglobin (HbA1c) from baseline. Other endpoints included proportion of participants with ≥0.5% HbA1c reduction and assessments of 2-hour postprandial glucose (PPG), weight, and urinary glucose excretion (UGE). RESULTS: From a mean baseline of 8.0% ± 0.8% (full study population), placebo-adjusted LSM HbA1c decreased by 0.3% (P = .07), 0.5% (P < .001) and 0.4% (P = .006) with sotagliflozin 75 mg, 200 mg and 400 mg, respectively, at week 12. In the placebo and sotagliflozin 75 mg, 200 mg and 400 mg groups, 33.3%, 37.1%, 80.0% and 65.7% of participants achieved an HbA1c reduction ≥0.5%. Placebo-adjusted PPG decreased by 22.2 mg/dL (P = .28), 28.7 mg/dL (P = .16) and 50.2 mg/dL (P = .013), UGE increased by 41.8 g/d (P = .006), 57.7 g/d (P < .001) and 70.5 g/d (P < .001), and weight decreased by 1.3 kg (P = .038), 2.4 kg (P < .001) and 2.6 kg (P < .001) with sotagliflozin 75 mg, 200 mg and 400 mg, respectively. One case of severe hypoglycaemia occurred in each sotagliflozin group and one case of diabetic ketoacidosis (DKA) occurred with sotagliflozin 400 mg. CONCLUSIONS: Combined with stable insulin doses, sotagliflozin 200 mg and 400 mg improved glycaemic control and weight in adults with T1D. Sotagliflozin 400 mg reduced PPG levels. UGE increased with all sotagliflozin doses. Rates of severe hypoglycaemia and DKA were low (NCT02459899).

Transcriptomic characterization of signaling pathways associated with osteoblastic differentiation of MC-3T3E1 cells.

Bone remodeling involves the coordinated actions of osteoclasts, which resorb the calcified bony matrix, and osteoblasts, which refill erosion pits created by osteoclasts to restore skeletal integrity and adapt to changes in mechanical load. Osteoblasts are derived from pluripotent mesenchymal stem cell precursors, which undergo differentiation under the influence of a host of local and environmental cues. To characterize the autocrine/paracrine signaling networks associated with osteoblast maturation and function, we performed gene network analysis using complementary "agnostic" DNA microarray and "targeted" NanoString nCounter datasets derived from murine MC3T3-E1 cells induced to undergo synchronized osteoblastic differentiation in vitro. Pairwise datasets representing changes in gene expression associated with growth arrest (day 2 to 5 in culture), differentiation (day 5 to 10 in culture), and osteoblast maturation (day 10 to 28 in culture) were analyzed using Ingenuity Systems Pathways Analysis to generate predictions about signaling pathway activity based on the temporal sequence of changes in target gene expression. Our data indicate that some pathways involved in osteoblast differentiation, e.g. Wnt/ß-catenin signaling, are most active early in the process, while others, e.g. TGFß/BMP, cytokine/JAK-STAT and TNFα/RANKL signaling, increase in activity as differentiation progresses. Collectively, these pathways contribute to the sequential expression of genes involved in the synthesis and mineralization of extracellular matrix. These results provide insight into the temporal coordination and complex interplay between signaling networks controlling gene expression during osteoblast differentiation. A more complete understanding of these processes may aid the discovery of novel methods to promote osteoblast development for the treatment of conditions characterized by low bone mineral density.

Translating in vitro ligand bias into in vivo efficacy.

It is increasingly apparent that ligand structure influences both the efficiency with which G protein-coupled receptors (GPCRs) engage their downstream effectors and the manner in which they are activated. Thus, 'biased' agonists, synthetic ligands whose intrinsic efficacy differs from the native ligand, afford a strategy for manipulating GPCR signaling in ways that promote beneficial signals while blocking potentially deleterious ones. Still, there are significant challenges in relating in vitro ligand efficacy, which is typically measured in heterologous expression systems, to the biological response in vivo, where the ligand is acting on natively expressed receptors and in the presence of the endogenous ligand. This is particularly true of arrestin pathway-selective 'biased' agonists. The type 1 parathyroid hormone receptor (PTH1R) is a case in point. Parathyroid hormone (PTH) is the principal physiological regulator of calcium homeostasis, and PTH1R expressed on cells of the osteoblast lineage are an established therapeutic target in osteoporosis. In vitro, PTH1R signaling is highly sensitive to ligand structure, and PTH analogs that affect the selectivity/kinetics of G protein coupling or that engage arrestin-dependent signaling mechanisms without activating heterotrimeric G proteins have been identified. In vivo, intermittent administration of conventional PTH analogs accelerates the rate of osteoblastic bone formation, largely through known cAMP-dependent mechanisms. Paradoxically, both intermittent and continuous administration of an arrestin pathway-selective PTH analog, which in vivo would be expected to antagonize endogenous PTH1R-cAMP signaling, also increases bone mass. Transcriptomic analysis of tissue from treated animals suggests that conventional and arrestin pathway-selective PTH1R ligands act in largely different ways, with the latter principally affecting pathways involved in the regulation of cell cycle, survival, and migration/cytoskeletal dynamics. Such multi-dimensional in vitro and in vivo analyses of ligand bias may provide insights into the physiological roles of non-canonical arrestin-mediated signaling pathways in vivo, and provide a conceptual framework for translating arrestin pathway-selective ligands into viable therapeutics.

Effects of Sotagliflozin Added to Insulin in Patients with Type 1 Diabetes.

BACKGROUND: In most patients with type 1 diabetes, adequate glycemic control is not achieved with insulin therapy alone. We evaluated the safety and efficacy of sotagliflozin, an oral inhibitor of sodium-glucose cotransporters 1 and 2, in combination with insulin treatment in patients with type 1 diabetes. METHODS: In this phase 3, double-blind trial, which was conducted at 133 centers worldwide, we randomly assigned 1402 patients with type 1 diabetes who were receiving treatment with any insulin therapy (pump or injections) to receive sotagliflozin (400 mg per day) or placebo for 24 weeks. The primary end point was a glycated hemoglobin level lower than 7.0% at week 24, with no episodes of severe hypoglycemia or diabetic ketoacidosis after randomization. Secondary end points included the change from baseline in glycated hemoglobin level, weight, systolic blood pressure, and mean daily bolus dose of insulin. RESULTS: A significantly larger proportion of patients in the sotagliflozin group than in the placebo group achieved the primary end point (200 of 699 patients [28.6%] vs. 107 of 703 [15.2%], P<0.001). The least-squares mean change from baseline was significantly greater in the sotagliflozin group than in the placebo group for glycated hemoglobin (difference, -0.46 percentage points), weight (-2.98 kg), systolic blood pressure (-3.5 mm Hg), and mean daily bolus dose of insulin (-2.8 units per day) (P≤0.002 for all comparisons). The rate of severe hypoglycemia was similar in the sotagliflozin group and the placebo group (3.0% [21 patients] and 2.4% [17], respectively). The rate of documented hypoglycemia with a blood glucose level of 55 mg per deciliter (3.1 mmol per liter) or below was significantly lower in the sotagliflozin group than in the placebo group. The rate of diabetic ketoacidosis was higher in the sotagliflozin group than in the placebo group (3.0% [21 patients] and 0.6% [4], respectively). CONCLUSIONS: Among patients with type 1 diabetes who were receiving insulin, the proportion of patients who achieved a glycated hemoglobin level lower than 7.0% with no severe hypoglycemia or diabetic ketoacidosis was larger in the group that received sotagliflozin than in the placebo group. However, the rate of diabetic ketoacidosis was higher in the sotagliflozin group. (Funded by Lexicon Pharmaceuticals; inTandem3 ClinicalTrials.gov number, NCT02531035 .).

Informatic deconvolution of biased GPCR signaling mechanisms from in vivo pharmacological experimentation.

Ligands possessing different physico-chemical structures productively interact with G protein-coupled receptors generating distinct downstream signaling events due to their abilities to activate/select idiosyncratic receptor entities ('receptorsomes') from the full spectrum of potential receptor partners. We have employed multiple novel informatic approaches to identify and characterize the in vivo transcriptomic signature of an arrestin-signaling biased ligand, [D-Trp(12),Tyr(34)]-bPTH(7-34), acting at the parathyroid hormone type 1 receptor (PTH1R), across six different murine tissues after chronic drug exposure. We are able to demonstrate that [D-Trp(12),Tyr(34)]-bPTH(7-34) elicits a distinctive arrestin-signaling focused transcriptomic response that is more coherently regulated, in an arrestin signaling-dependent manner, across more tissues than that of the pluripotent endogenous PTH1R ligand, hPTH(1-34). This arrestin-focused response signature is strongly linked with the transcriptional regulation of cell growth and development. Our informatic deconvolution of a conserved arrestin-dependent transcriptomic signature from wild type mice demonstrates a conceptual framework within which the in vivo outcomes of biased receptor signaling may be further investigated or predicted.

Delineation of a conserved arrestin-biased signaling repertoire in vivo.

Biased G protein-coupled receptor agonists engender a restricted repertoire of downstream events from their cognate receptors, permitting them to produce mixed agonist-antagonist effects in vivo. While this opens the possibility of novel therapeutics, it complicates rational drug design, since the in vivo response to a biased agonist cannot be reliably predicted from its in cellula efficacy. We have employed novel informatic approaches to characterize the in vivo transcriptomic signature of the arrestin pathway-selective parathyroid hormone analog [d-Trp(12), Tyr(34)]bovine PTH(7-34) in six different murine tissues after chronic drug exposure. We find that [d-Trp(12), Tyr(34)]bovine PTH(7-34) elicits a distinctive arrestin-signaling focused transcriptomic response that is more coherently regulated across tissues than that of the pluripotent agonist, human PTH(1-34). This arrestin-focused network is closely associated with transcriptional control of cell growth and development. Our demonstration of a conserved arrestin-dependent transcriptomic signature suggests a framework within which the in vivo outcomes of arrestin-biased signaling may be generalized.

Arrestins in bone.

Parathyroid hormone (PTH) is the principle regulator of calcium-phosphorus metabolism and bone turnover. Because of its central role in bone remodeling, recombinant human PTH (i.e., Forteo®; PTH(1-34)) has been developed for the treatment of osteoporosis. PTH(1-34) acts principally through the type I PTH/PTH-related peptide receptor (PTH1R), a classic seven transmembrane G protein-coupled receptor (GPCR). Intermittent treatment with PTH(1-34) promotes osteoblast and osteoclast recruitment through activation of PTH1R with resultant net bone gain. Recent studies have demonstrated that the complex metabolic effects induced by PTH1R stimulation are not entirely a consequence of conventional GPCR signaling. ß-Arrestins, in addition to their desensitizing actions, also serve as multifunctional scaffolding proteins linking the PTH1R to signaling molecules independent of classic G protein-mediated second messenger-dependent pathways. In vitro, D-Trp(12), Tyr(34)-bPTH(7-34) [bPTH(7-34)], a ß-arrestin-selective biased agonist for the PTH1R, antagonizes G protein signaling but activates arrestin-dependent signaling. In vivo, intermittent administration of bPTH(7-34) to mice induces anabolic bone formation independent of classic G protein-coupled signaling mechanisms. While both the conventional PTH1R agonists, PTH(1-34) and bPTH(7-34), stimulate anabolic bone formation in mice, the latter does not induce hypercalcemia nor does it increase markers of bone resorption. This newly recognized ability of ß-arrestins to serve as signal transducers for the PTH1R independent of classic GPCR signaling represents a novel paradigm with therapeutic potential. Exploitation of ß-arrestin-biased agonism may offer therapeutic benefit for the treatment of metabolic bone diseases such as osteoporosis with an improved side effect profile.

ß-arrestin-selective G protein-coupled receptor agonists engender unique biological efficacy in vivo.

Biased G protein-coupled receptor agonists are orthosteric ligands that possess pathway-selective efficacy, activating or inhibiting only a subset of the signaling repertoire of their cognate receptors. In vitro, D-Trp(12),Tyr(34)-bPTH(7-34) [bPTH(7-34)], a biased agonist for the type 1 PTH receptor, antagonizes receptor-G protein coupling but activates arrestin-dependent signaling. In vivo, both bPTH(7-34) and the conventional agonist hPTH(1-34) stimulate anabolic bone formation. To understand how two PTH receptor ligands with markedly different in vitro efficacy could elicit similar in vivo responses, we analyzed transcriptional profiles from calvarial bone of mice treated for 8 wk with vehicle, bPTH(7-34) or hPTH(1-34). Treatment of wild-type mice with bPTH(7-34) primarily affected pathways that promote expansion of the osteoblast pool, notably cell cycle regulation, cell survival, and migration. These responses were absent in ß-arrestin2-null mice, identifying them as downstream targets of ß-arrestin2-mediated signaling. In contrast, hPTH(1-34) primarily affected pathways classically associated with enhanced bone formation, including collagen synthesis and matrix mineralization. hPTH(1-34) actions were less dependent on ß-arrestin2, as might be expected of a ligand capable of G protein activation. In vitro, bPTH(7-34) slowed the rate of preosteoblast proliferation, enhanced osteoblast survival when exposed to an apoptotic stimulus, and stimulated cell migration in wild-type, but not ß-arrestin2-null, calvarial osteoblasts. These results suggest that bPTH(7-34) and hPTH(1-34) affect bone mass in vivo through predominantly separate genomic mechanisms created by largely distinct receptor-signaling networks and demonstrate that functional selectivity can be exploited to change the quality of G protein-coupled receptor efficacy.

The cytoskeletal regulatory scaffold protein GIT2 modulates mesenchymal stem cell differentiation and osteoblastogenesis.

G protein-coupled receptor kinase interacting protein 2 (GIT2) is a signaling scaffold protein involved in the regulation of cytoskeletal structure, membrane trafficking, and G protein-coupled receptor internalization. Since dynamic cytoskeletal reorganization plays key roles both in osteoblast differentiation and in the maintenance of osteoclast polarity during bone resorption, we hypothesized that skeletal physiology would be altered in GIT2(-/-) mice. We found that adult GIT2(-/-) mice have decreased bone mineral density and bone volume in both the trabecular and cortical compartments. This osteopenia was associated with decreased numbers of mature osteoblasts, diminished osteoblastic activity, and increased marrow adiposity, suggesting a defect in osteoblast maturation. In vitro, mesenchymal stem cells derived from GIT2(-/-) mice exhibited impaired differentiation into osteoblasts and increased adipocyte differentiation, consistent with a role for GIT2 in mesenchymal stem cell fate determination. Despite elevated osteoclast inducing cytokines and osteoclast numbers, GIT2(-/-) mice also exhibit impaired bone resorption, consistent with a further role for GIT2 in regulating osteoclast function. Collectively, these findings underscore the importance of the cytoskeleton in both osteoblast and osteoclast function and demonstrate that GIT2 plays essential roles in skeletal metabolism, affecting both bone formation and bone resorption in vivo.

The oxysterol, 27-hydroxycholesterol, links cholesterol metabolism to bone homeostasis through its actions on the estrogen and liver X receptors.

Osteoporosis and age-related bone loss are important public health concerns. Therefore, there is a high level of interest in the development of medical interventions and lifestyle changes that reduce the incidence of osteoporosis and age-related bone loss. Decreased bone mineral density is associated with high cholesterol, and patients on statins have increased bone mineral densities, strongly implicating cholesterol as a negative regulator of bone homeostasis. In this study, using both molecular and pharmacological approaches, we have been able to demonstrate that the primary cholesterol metabolite, 27-hydroxycholesterol, through its actions on both estrogen receptors and liver X receptors, decreases osteoblast differentiation and enhances osteoclastogenesis, resulting in increased bone resorbtion in mice. Induction of the short heterodimer partner protein by estrogens in osteoblasts can attenuate the liver X receptor-mediated actions of 27-hydroxycholesterol in bone. These data establish a mechanistic link between cholesterol and bone quality, highlight an unexpected target of estrogens in osteoblasts, and define a signaling axis, the therapeutic exploitation of which is likely to yield novel antiosteoporotic drugs.

Refining efficacy: exploiting functional selectivity for drug discovery.

Early models of G protein-coupled receptor (GPCR) activation envisioned the receptor in equilibrium between unique "off" and "on" states, wherein ligand binding affected signaling by increasing or decreasing the fraction of receptors in the active conformation. It is now apparent that GPCRs spontaneously sample multiple conformations, any number of which may couple to one or more downstream effectors. Such "multistate" models imply that the receptor-ligand complex, not the receptor alone, defines which active receptor conformations predominate. "Functional selectivity" refers to the ability of a ligand to activate only a subset of its receptor's signaling repertoire. There are now numerous examples of ligands that "bias" receptor coupling between different G protein pools and non-G protein effectors such as arrestins. The type 1 parathyroid hormone receptor (PTH(1)R) is a particularly informative example, not only because of the range of biased effects that have been produced, but also because the actions of many of these ligands have been characterized in vivo. Biased PTH(1)R ligands can selectively couple the PTH(1)R to G(s) or G(q/11) pathways, with or without activating arrestin-dependent receptor desensitization and signaling. These reagents have provided insight into the contribution of different signaling pathways to PTH action in vivo and suggest it may be possible to exploit ligand bias to uncouple the anabolic effects of PTH(1)R from its catabolic and calcitropic effects. Whereas conventional agonists and antagonists only modulate the quantity of efficacy, functionally selective ligands qualitatively change GPCR signaling, offering the prospect of drugs with improved therapeutic efficacy or reduced side effects.

ß-arrestin-biased agonism at the parathyroid hormone receptor uncouples bone formation from bone resorption.

Parathyroid hormone (PTH) is a principle regulator of bone and calcium metabolism and PTH analogs hold great promise as a therapy for metabolic bone diseases such as osteoporosis. PTH acts principally through the type IPTH/PTH-related peptide receptor (PTH1R), a G protein coupled receptor (GPCR). GPCRs are a family of seven transmembrane cell surface receptors that share conserved structural, functional, and regulatory properties. Recent studies demonstrate that the complex metabolic effects induced by PTH1R stimulation are not entirely a consequence of conventional GPCR signaling. ß-arrestins, in addition to their GPCR desensitizing actions, also serve as multifunctional scaffolding proteins linking the PTH1R to signaling molecules independent of the classic G protein coupled second messenger-dependent pathways. In vitro, D-Trp(12),Tyr(34)-bPTH(7-34) (PTH-ßarr), a ß-arrestin selective biased agonist for the PTH1R, antagonizes receptor-G protein coupling but activates arrestin-dependent signaling. In vivo, intermittent administration of, PTH-ßarr to mice, induces anabolic bone formation, completely independent of classic G protein-coupled signaling mechanisms. While both PTH-ßarr and the conventional agonist PTH(1-34) stimulate anabolic bone formation in mice, unlike PTH(1-34), which activates G protein coupling, PTH-ßarr does not induce hypercalcemia or increase markers of bone resorption. This newly recognized ability of ß-arrestins to serve as signal transducers for the PTH1R represents an innovative paradigm of receptor signaling which can be targeted to induce a subset of physiologic responses in bone. Exploitation of ß-arrestin biased agonism may offer therapeutic benefit for the treatment of metabolic bone diseases such as osteoporosis.

'Biasing' the parathyroid hormone receptor: a novel anabolic approach to increasing bone mass?

'Functional selectivity' refers to the ability of a ligand to activate and/or inhibit only a subset of the signals capable of emanating from its cognate G-protein-coupled receptor (GPCR). Whereas conventional GPCR agonism and antagonism can be viewed as modulating the quantity of efficacy, functionally selective or 'biased' ligands qualitatively change the nature of information flow across the plasma membrane, raising the prospect of drugs with improved therapeutic efficacy or reduced side effects. Nonetheless, there is little experimental evidence that biased ligands offer advantages over conventional agonists/antagonists in vivo. Recent work with the type I parathyroid hormone receptor (PTH(1) R) suggests that biased ligands that selectively activate G-protein-independent arrestin-mediated signalling pathways may hold promise in the treatment of osteoporosis. Parathyroid hormone (PTH) is a principle regulator of bone and calcium metabolism. In bone, PTH exerts complex effects; promoting new bone formation through direct actions on osteoblasts while simultaneously stimulating bone loss through indirect activation of osteoclastic bone resorption. Although the conventional PTH(1) R agonist teriparatide, PTH(1-34), is effective in the treatment of osteoporosis, its utility is limited by its bone-resorptive effects and propensity to promote hypercalcaemia/hypercalcuria. In contrast, d-Trp(12) ,Tyr(34) -bPTH(7-34) (PTH-ßarr), an arrestin pathway-selective agonist for the PTH(1) R, induces anabolic bone formation independent of classic G-protein-coupled signalling mechanisms. Unlike PTH(1-34), PTH-ßarr appears to 'uncouple' the anabolic effects of PTH(1) R activation from its catabolic and calcitropic effects. Such findings offer evidence that arrestin pathway-selective GPCR agonists can elicit potentially beneficial effects in vivo that cannot be achieved using conventional agonist or antagonist ligands.

Regulator of G protein signaling 5 is highly expressed in parathyroid tumors and inhibits signaling by the calcium-sensing receptor.

The molecular mechanisms responsible for aberrant calcium signaling in parathyroid disease are poorly understood. The loss of appropriate calcium-responsive modulation of PTH secretion observed in parathyroid disease is commonly attributed to decreased expression of the calcium-sensing receptor (CaSR), a G protein-coupled receptor. However, CaSR expression is highly variable in parathyroid adenomas, and the lack of correlation between CaSR abundance and calcium-responsive PTH kinetics indicates that mechanisms independent of CaSR expression may contribute to aberrant calcium sensing in parathyroid disease. To gain a better understanding of parathyroid tumors and the molecular determinants that drive parathyroid adenoma development, we performed gene expression profiling on a panel of 64 normal and neoplastic parathyroid tissues. The microarray data revealed high-level expression of genes known to be involved in parathyroid biology (PTH, VDR, CGA, CaSR, and GCM2). Moreover, our screen identified regulator of G protein signaling 5 (RGS5) as a candidate inhibitor of CaSR signaling. We confirmed RGS5 to be highly expressed in parathyroid adenomas relative to matched-pair normal glands. Transient expression of RGS5 in cells stably expressing CaSR resulted in dose-dependent abrogation of calcium-stimulated inositol trisphosphate production and ERK1/2 phosphorylation. Furthermore, we found that RGS5-nullizygous mice display reduced plasma PTH levels, an outcome consistent with attenuated opposition to CaSR activity. Collectively, these data suggest that RGS5 can act as a physiological regulator of calcium sensing by CaSR in the parathyroid gland. The abnormally elevated expression of RGS5 observed in parathyroid adenomas could thus represent a novel mechanism of CaSR desensitization in patients with primary hyperparathyroidism.

Osteoporosis in lung transplant candidates compared to matched healthy controls.

PURPOSE: Advanced lung disease increases the risk for diminished bone mineral density (BMD). The prevalence and severity of osteoporosis in lung transplant candidates is unclear. METHODS: We retrospectively evaluated BMD of subjects screened for lung transplant at our institution. Observed prevalence of osteoporosis and osteopenia within our cohort was compared to the expected prevalence of each from the Third National Health and Nutrition Examination Survey (NHANES III) data matched for age, gender, and race. Lateral chest radiographs were evaluated for vertebral fractures. RESULTS: High prevalence rates of osteoporosis (37%) and combined osteoporosis/osteopenia (86%) were observed. Subjects with pulmonary fibrosis had higher BMD and T-scores compared to all other subgroups. All subjects within the cohort had a higher observed combined rate of osteoporosis/osteopenia at all bone sites compared to expected rates from healthy, matched controls. Vertebral fractures were present in 23% of subjects but did not correlate with BMD or the diagnosis of osteoporosis. CONCLUSIONS: Abnormal BMD was prevalent in most pre-lung transplant subjects, with striking differences noted in comparison with a healthy, matched cohort. Lateral chest radiographs in combination with BMD data give a more complete picture of bone abnormalities. Osteoporosis screening prior to lung transplantation should be performed to identify high-risk subjects for fracture and allow for intervention.

Growth hormone mitigates against lethal irradiation and enhances hematologic and immune recovery in mice and nonhuman primates.

Medications that can mitigate against radiation injury are limited. In this study, we investigated the ability of recombinant human growth hormone (rhGH) to mitigate against radiation injury in mice and nonhuman primates. BALB/c mice were irradiated with 7.5 Gy and treated post-irradiation with rhGH intravenously at a once daily dose of 20 microg/dose for 35 days. rhGH protected 17 out of 28 mice (60.7%) from lethal irradiation while only 3 out of 28 mice (10.7%) survived in the saline control group. A shorter course of 5 days of rhGH post-irradiation produced similar results. Compared with the saline control group, treatment with rhGH on irradiated BALB/c mice significantly accelerated overall hematopoietic recovery. Specifically, the recovery of total white cells, CD4 and CD8 T cell subsets, B cells, NK cells and especially platelets post radiation exposure were significantly accelerated in the rhGH-treated mice. Moreover, treatment with rhGH increased the frequency of hematopoietic stem/progenitor cells as measured by flow cytometry and colony forming unit assays in bone marrow harvested at day 14 after irradiation, suggesting the effects of rhGH are at the hematopoietic stem/progenitor level. rhGH mediated the hematopoietic effects primarily through their niches. Similar data with rhGH were also observed following 2 Gy sublethal irradiation of nonhuman primates. Our data demonstrate that rhGH promotes hematopoietic engraftment and immune recovery post the exposure of ionizing radiation and mitigates against the mortality from lethal irradiation even when administered after exposure.

The endogenous selective estrogen receptor modulator 27-hydroxycholesterol is a negative regulator of bone homeostasis.

Osteoporosis is an important clinical problem, affecting more than 50% of people over age 50 yr. Estrogen signaling is critical for maintaining proper bone density, and the identification of an endogenous selective estrogen receptor (ER) modulator, 27-hydroxycholesterol (27HC), suggests a mechanism by which nutritional/metabolic status can influence bone biology. With its levels directly correlated with cholesterol, a new possibility emerges wherein 27HC links estrogen and cholesterol signaling to bone homeostasis. In these studies, we found that increasing concentrations of 27HC, both by genetic and pharmacological means, led to decreased bone mineral density that was associated with decreased bone formation and increased bone resorption. Upon manipulation of endogenous estrogen levels, many of the responses to elevated 27HC were altered in such a way as to implicate ER as a likely mediator. In a model of postmenopausal bone loss, some pathologies associated with elevated 27HC were exacerbated by the absence of endogenous estrogens, suggesting that 27HC may act both in concert with and independently from classic ER signaling. These data provide evidence for interactions between estrogen signaling, cholesterol and metabolic disease, and osteoporosis. Patients with high cholesterol likely also have higher than average 27HC, perhaps putting them at a higher risk for bone loss and fracture. More studies are warranted to fully elucidate the mechanism of action of 27HC in bone and to identify ways to modulate this pathway therapeutically.

Beyond desensitization: physiological relevance of arrestin-dependent signaling.

Heptahelical G protein-coupled receptors are the most diverse and therapeutically important family of receptors in the human genome. Ligand binding activates heterotrimeric G proteins that transmit intracellular signals by regulating effector enzymes or ion channels. G protein signaling is terminated, in large part, by arrestin binding, which uncouples the receptor and G protein and targets the receptor for internalization. It is clear, however, that heptahelical receptor signaling does not end with desensitization. Arrestins bind a host of catalytically active proteins and serve as ligand-regulated scaffolds that recruit protein and lipid kinase, phosphatase, phosphodiesterase, and ubiquitin ligase activity into the receptor-arrestin complex. Although many of these arrestin-bound effectors serve to modulate G protein signaling, degrading second messengers and regulating endocytosis and trafficking, other signals seem to extend beyond the receptor-arrestin complex to regulate such processes as protein translation and gene transcription. Although these findings have led to a re-envisioning of heptahelical receptor signaling, little is known about the physiological roles of arrestin-dependent signaling. In vivo, the duality of arrestin function makes it difficult to dissociate the consequences of arrestin-dependent desensitization from those that might be ascribed to arrestin-mediated signaling. Nonetheless, recent evidence generated using arrestin knockouts, G protein-uncoupled receptor mutants, and arrestin pathway-selective "biased agonists" is beginning to reveal that arrestin signaling plays important roles in the retina, central nervous system, cardiovascular system, bone remodeling, immune system, and cancer. Understanding the signaling roles of arrestins may foster the development of pathway-selective drugs that exploit these pathways for therapeutic benefit.

Inhibition of WNT signaling by G protein-coupled receptor (GPCR) kinase 2 (GRK2).

Activation of Wnt signaling pathways causes release and stabilization of the transcription regulator beta-catenin from a destruction complex composed of axin and the adenomatous polyposis coli (APC) protein (canonical signaling pathway). Assembly of this complex is facilitated by a protein-protein interaction between APC and a regulator of G protein signaling (RGS) domain in axin. Because G protein-coupled receptor kinase 2 (GRK2) has a RGS domain that is closely related to the RGS domain in axin, we determined whether GRK2 regulated canonical signaling. We found that GRK2 inhibited Wnt1-induced activation of a reporter construct as well as reduced Wnt3a-dependent stabilization and nuclear translocation of beta-catenin. GRK2 enzymatic activity was required for this negative regulatory effect, and depletion of endogenous GRK2 using small interfering RNA enhanced canonical signaling. GRK2-dependent inhibition of canonical signaling is relevant to osteoblast (OB) biology because overexpression of GRK2 attenuated Wnt/beta-catenin signaling in calvarial OBs. Coimmunoprecipitation studies found that: 1) GRK2 bound APC; 2) The GRK2-APC interaction was promoted by GRK2 enzymatic activity; and 3) Deletion of the RGS domain in GRK2 prevented both the GRK2-APC interaction and GRK2-dependent inhibition of canonical signaling. These data suggest that: 1) GRK2 negatively regulates Wnt signaling; 2) GRK2-dependent inhibition of canonical signaling requires a protein-protein interaction between the RGS domain in GRK2 and APC; and 3) Enzymatic activity promotes the GRK2-APC interaction and is required for the negative regulatory effect on canonical signaling. We speculate that inhibiting GRK2 activity in bone-forming OBs might be a useful therapeutic strategy for increasing bone mass.

A beta-arrestin-biased agonist of the parathyroid hormone receptor (PTH1R) promotes bone formation independent of G protein activation.

About 40% of the therapeutic agents in use today exert their effects through seven-transmembrane receptors (7TMRs). When activated by ligands, these receptors trigger two pathways that independently transduce signals to the cell: one through heterotrimeric GTP-binding proteins (G proteins) and one through beta-arrestins; so-called biased agonists can selectively activate these distinct pathways. Here, we investigate selective activation of these pathways through the use of a biased agonist for the type 1 parathyroid hormone (PTH)-PTH-related protein receptor (PTH1R), (D-Trp(12),Tyr(34))-PTH(7-34) (PTH-betaarr), which activates beta-arrestin but not classic G protein signaling. In mice, PTH-betaarr induces anabolic bone formation, as does the nonselective agonist PTH(1-34), which activates both mechanisms. In beta-arrestin2-null mice, the increase in bone mineral density evoked by PTH(1-34) is attenuated and that stimulated by PTH-betaarr is ablated. The beta-arrestin2-dependent pathway contributes primarily to trabecular bone formation and does not stimulate bone resorption. These results show that a biased agonist selective for the beta-arrestin pathway can elicit a response in vivo distinct from that elicited by nonselective agonists. Ligands with these properties may form the basis for improved 7TMR-directed pharmacologic agents with enhanced therapeutic specificity.