G-CSF Receptor Deletion Amplifies Cortical Bone Dysfunction in Mice With STAT3 Hyperactivation in Osteocytes.

Bone strength is determined by the structure and composition of its thickened outer shell (cortical bone), yet the mechanisms controlling cortical consolidation are poorly understood. Cortical bone maturation depends on SOCS3-mediated suppression of IL-6 cytokine-induced STAT3 phosphorylation in osteocytes, the cellular network embedded in bone matrix. Because SOCS3 also suppresses granulocyte-colony-stimulating factor receptor (G-CSFR) signaling, we here tested whether global G-CSFR (Csf3r) ablation altereed bone structure in male and female mice lacking SOCS3 in osteocytes, (Dmp1Cre :Socs3f/f mice). Dmp1Cre :Socs3f/f :Csf3r-/- mice were generated by crossing Dmp1Cre :Socs3f/f mice with Csf3r-/- mice. Although G-CSFR is not expressed in osteocytes, Csf3r deletion further delayed cortical consolidation in Dmp1Cre :Socs3f/f mice. Micro-CT images revealed extensive, highly porous low-density bone, with little true cortex in the diaphysis, even at 26 weeks of age; including more low-density bone and less high-density bone in Dmp1Cre :Socs3f/f :Csf3r-/- mice than controls. By histology, the area where cortical bone would normally be found contained immature compressed trabecular bone in Dmp1Cre :Socs3f/f :Csf3r-/- mice and greater than normal levels of intracortical osteoclasts, extensive new woven bone formation, and the presence of more intracortical blood vessels than the already high levels observed in Dmp1Cre :Socs3f/f controls. qRT-PCR of cortical bone from Dmp1Cre :Socs3f/f :Csf3r-/- mice also showed more than a doubling of mRNA levels for osteoclasts, osteoblasts, RANKL, and angiogenesis markers. The further delay in cortical bone maturation was associated with significantly more phospho-STAT1 and phospho-STAT3-positive osteocytes, and a threefold increase in STAT1 and STAT3 target gene mRNA levels, suggesting G-CSFR deletion further increases STAT signaling beyond that of Dmp1Cre :Socs3f/f bone. G-CSFR deficiency therefore promotes STAT1/3 signaling in osteocytes, and when SOCS3 negative feedback is absent, elevated local angiogenesis, bone resorption, and bone formation delays cortical bone consolidation. This points to a critical role of G-CSF in replacing condensed trabecular bone with lamellar bone during cortical bone formation. © 2022 American Society for Bone and Mineral Research (ASBMR).

The effect of carbamazepine on bone structure and strength in control and osteogenesis imperfecta (Col1a2 +/p.G610C ) mice.

The inherited brittle bone disease osteogenesis imperfecta (OI) is commonly caused by COL1A1 and COL1A2 mutations that disrupt the collagen I triple helix. This causes intracellular endoplasmic reticulum (ER) retention of the misfolded collagen and can result in a pathological ER stress response. A therapeutic approach to reduce this toxic mutant load could be to stimulate mutant collagen degradation by manipulating autophagy and/or ER-associated degradation. Since carbamazepine (CBZ) both stimulates autophagy of misfolded collagen X and improves skeletal pathology in a metaphyseal chondrodysplasia model, we tested the effect of CBZ on bone structure and strength in 3-week-old male OI Col1a2 +/p.G610C and control mice. Treatment for 3 or 6 weeks with CBZ, at the dose effective in metaphyseal chondrodysplasia, provided no therapeutic benefit to Col1a2 +/p.G610C mouse bone structure, strength or composition, measured by micro-computed tomography, three point bending tests and Fourier-transform infrared microspectroscopy. In control mice, however, CBZ treatment for 6 weeks impaired femur growth and led to lower femoral cortical and trabecular bone mass. These data, showing the negative impact of CBZ treatment on the developing mouse bones, raise important issues which must be considered in any human clinical applications of CBZ in growing individuals.

Structural biology of cell surface receptors implicated in Alzheimer's disease.

Alzheimer's disease is a common and devastating age-related disease with no effective disease-modifying treatments. Human genetics has implicated a wide range of cell surface receptors as playing a role in the disease, many of which are involved in the production or clearance of neurotoxins in the brain. Amyloid precursor protein, a membrane-bound signaling molecule, is at the very heart of the disease: hereditary mutations in its gene are associated with a greatly increased risk of getting the disease. A proteolytic breakdown product of amyloid precursor protein, the neurotoxic Aß peptide, has been the target for many drug discovery efforts. Antibodies have been designed to target Aß production with some success, although they have not proved efficacious in clinical trials with regards to cognitive benefits to date. Many of the recently identified genes associated with late-onset Alzheimer's disease risk are integral to the innate immune system. Some of these genes code for microglial proteins, such as the strongest genetic risk factor for the disease, namely APOE, and the cell surface receptors CD33 and TREM2 which are involved in clearance of the Aß peptide from the brain. In this review, we show how structural biology has provided key insights into the normal functioning of these cell surface receptors and provided a framework for developing novel treatments to combat Alzheimer's disease.

Characterization of Skeletal Phenotype and Associated Mechanisms With Chronic Intestinal Inflammation in the Winnie Mouse Model of Spontaneous Chronic Colitis.

BACKGROUND: Osteoporosis is a common extraintestinal manifestation of inflammatory bowel disease (IBD). However, studies have been scarce, mainly because of the lack of an appropriate animal model of colitis-associated bone loss. In this study, we aimed to decipher skeletal manifestations in the Winnie mouse model of spontaneous chronic colitis, which carries a MUC2 gene mutation and closely replicates ulcerative colitis. In our study, Winnie mice, prior to the colitis onset at 6 weeks old and progression at 14 and 24 weeks old, were compared with age-matched C57BL/6 controls. We studied several possible mechanisms involved in colitis-associated bone loss. METHODS: We assessed for bone quality (eg, microcomputed tomography [micro-CT], static and dynamic histomorphometry, 3-point bending, and ex vivo bone marrow analysis) and associated mechanisms (eg, electrochemical recordings for gut-derived serotonin levels, real-time polymerase chain reaction [qRT-PCR], double immunofluorescence microscopy, intestinal inflammation levels by lipocalin-2 assay, serum levels of calcium, phosphorus, and vitamin D) from Winnie (6-24 weeks) and age-matched C57BL6 mice. RESULTS: Deterioration in trabecular and cortical bone microarchitecture, reductions in bone formation, mineral apposition rate, bone volume/total volume, osteoid volume/bone surface, and bone strength were observed in Winnie mice compared with controls. Decreased osteoblast and increased osteoclast numbers were prominent in Winnie mice compared with controls. Upregulation of 5-HTR1B gene and increased association of FOXO1 with ATF4 complex were identified as associated mechanisms concomitant to overt inflammation and high levels of gut-derived serotonin in 14-week and 24-week Winnie mice. CONCLUSIONS: Skeletal phenotype of the Winnie mouse model of spontaneous chronic colitis closely represents manifestations of IBD-associated osteoporosis/osteopenia. The onset and progression of intestinal inflammation are associated with increased gut-derived serotonin level, increased bone resorption, and decreased bone formation.

Dmp1Cre-directed knockdown of parathyroid hormone-related protein (PTHrP) in murine decidua is associated with a life-long increase in bone mass, width, and strength in male progeny.

Parathyroid hormone-related protein (PTHrP, gene name Pthlh) is a pleiotropic regulator of tissue homeostasis. In bone, Dmp1Cre-targeted PTHrP deletion in osteocytes causes osteopenia and impaired cortical strength. We report here that this outcome depends on parental genotype. In contrast to our previous report using mice bred from heterozygous (flox/wild type) Dmp1Cre.Pthlhf/w parents, adult (16-week-old and 26-week-old) flox/flox (f/f) Dmp1Cre.Pthlhf/f mice from homozygous parents (Dmp1Cre.Pthlhf/f(hom) ) have stronger bones, with 40% more trabecular bone mass and 30% greater femoral width than controls. This greater bone size was observed in Dmp1Cre.Pthlhf/f(hom) mice as early as 12 days of age, when greater bone width was also found in male and female Dmp1Cre.Pthlhf/f(hom) mice compared to controls, but not in gene-matched mice from heterozygous parents. This suggested a maternal influence on skeletal size prior to weaning. Although Dmp1Cre has previously been reported to cause gene recombination in mammary gland, milk PTHrP protein levels were normal. The wide-bone phenotype was also noted in utero: Dmp1Cre.Pthlhf/f(hom) embryonic femurs were more mineralized and wider than controls. Closer examination revealed that Dmp1Cre caused PTHrP recombination in placenta, and in the maternal-derived decidual layer that resides between the placenta and the uterus. Decidua from mothers of Dmp1Cre.Pthlhf/f(hom) mice also exhibited lower PTHrP levels by immunohistochemistry and were smaller than controls. We conclude that Dmp1Cre leads to gene recombination in decidua, and that decidual PTHrP might, through an influence on decidual cells, limit embryonic bone radial growth. This suggests a maternal-derived developmental origin of adult bone strength. © 2021 American Society for Bone and Mineral Research (ASBMR).

Cortical bone maturation in mice requires SOCS3 suppression of gp130/STAT3 signalling in osteocytes.

Bone strength is determined by its dense cortical shell, generated by unknown mechanisms. Here we use the Dmp1Cre:Socs3f/f mouse, with delayed cortical bone consolidation, to characterise cortical maturation and identify control signals. We show that cortical maturation requires a reduction in cortical porosity, and a transition from low to high density bone, which continues even after cortical shape is established. Both processes were delayed in Dmp1Cre:Socs3f/f mice. SOCS3 (suppressor of cytokine signalling 3) inhibits signalling by leptin, G-CSF, and IL-6 family cytokines (gp130). In Dmp1Cre:Socs3f/f bone, STAT3 phosphorylation was prolonged in response to gp130-signalling cytokines, but not G-CSF or leptin. Deletion of gp130 in Dmp1Cre:Socs3f/f mice suppressed STAT3 phosphorylation in osteocytes and osteoclastic resorption within cortical bone, leading to rescue of the corticalisation defect, and restoration of compromised bone strength. We conclude that cortical bone development includes both pore closure and accumulation of high density bone, and that these processes require suppression of gp130-STAT3 signalling in osteocytes.

Small Molecule Binding to Alzheimer Risk Factor CD33 Promotes Aß Phagocytosis.

Polymorphism in the microglial receptor CD33 gene has been linked to late-onset Alzheimer disease (AD), and reduced expression of the CD33 sialic acid-binding domain confers protection. Thus, CD33 inhibition might be an effective therapy against disease progression. Progress toward discovery of selective CD33 inhibitors has been hampered by the absence of an atomic resolution structure. We report here the crystal structures of CD33 alone and bound to a subtype-selective sialic acid mimetic called P22 and use them to identify key binding residues by site-directed mutagenesis and binding assays to reveal the molecular basis for its selectivity toward sialylated glycoproteins and glycolipids. We show that P22, when presented on microparticles, increases uptake of the toxic AD peptide, amyloid-ß (Aß), into microglial cells. Thus, the sialic acid-binding site on CD33 is a promising pharmacophore for developing therapeutics that promote clearance of the Aß peptide that is thought to cause AD.

IL-6 exhibits both cis- and trans-signaling in osteocytes and osteoblasts, but only trans-signaling promotes bone formation and osteoclastogenesis.

Interleukin 6 (IL-6) supports development of bone-resorbing osteoclasts by acting early in the osteoblast lineage via membrane-bound (cis) or soluble (trans) receptors. Here, we investigated how IL-6 signals and modifies gene expression in differentiated osteoblasts and osteocytes and determined whether these activities can promote bone formation or support osteoclastogenesis. Moreover, we used a genetically altered mouse with circulating levels of the pharmacological IL-6 trans-signaling inhibitor sgp130-Fc to determine whether IL-6 trans-signaling is required for normal bone growth and remodeling. We found that IL-6 increases suppressor of cytokine signaling 3 (Socs3) and CCAAT enhancer-binding protein δ (Cebpd) mRNA levels and promotes signal transducer and activator of transcription 3 (STAT3) phosphorylation by both cis- and trans-signaling in cultured osteocytes. In contrast, RANKL (Tnfsf11) mRNA levels were elevated only by trans-signaling. Furthermore, we observed soluble IL-6 receptor release and ADAM metallopeptidase domain 17 (ADAM17) sheddase expression by osteocytes. Despite the observation that IL-6 cis-signaling occurs, IL-6 stimulated bone formation in vivo only via trans-signaling. Although IL-6 stimulated RANKL (Tnfsf11) mRNA in osteocytes, these cells did not support osteoclast formation in response to IL-6 alone; binucleated TRAP+ cells formed, and only in response to trans-signaling. Finally, pharmacological, sgp130-Fc-mediated inhibition of IL-6 trans-signaling did not impair bone growth or remodeling unless mice had circulating sgp130-Fc levels > 10 µg/ml. At those levels, osteopenia and impaired bone growth occurred, reducing bone strength. We conclude that high sgp130-Fc levels may have detrimental off-target effects on the skeleton.

Isolation, Purification, Generation, and Culture of Osteocytes.

Osteocytes reside within bone matrix and produce both paracrine and endocrine factors that influence the skeleton and other tissues. Despite their abundance and physiological importance, osteocytes have been difficult to study in vitro because they are difficult to extract and purify, and do not retain their phenotype in standard culture conditions. However, new techniques for this purpose are emerging. This chapter will describe three methods we use to study osteocytes: (1) isolating and purifying primary osteocytes from murine bone, with and without hematopoietic-lineage depletion, (2) differentiating cultured osteoblasts (or osteoblast cell lines) until they reach a stage of osteocytic gene expression, and (3) using the Ocy454 osteocyte-like cell line.

Adolescent Inhalant Abuse Results in Adrenal Dysfunction and a Hypermetabolic Phenotype with Persistent Growth Impairments.

BACKGROUND/AIMS: Abuse of toluene products (e.g., glue-sniffing) primarily occurs during adolescence and has been associated with appetite suppression and weight impairments. However, the metabolic phenotype arising from adolescent inhalant abuse has never been fully characterised, and its persistence during abstinence and underlying mechanisms remain unknown. METHODS: Adolescent male Wistar rats (post-natal day 27) were exposed to inhaled toluene (10,000 ppm) (n = 32) or air (n = 48) for 1 h/day, 3 days/week for 4 weeks, followed by 4 weeks of abstinence. Twenty air rats were pair-fed to the toluene group, to differentiate the direct effects of toluene from under-nutrition. Food intake, weight, and growth were monitored. Metabolic hormones were measured after exposure and abstinence periods. Energy expenditure was measured using indirect calorimetry. Adrenal function was assessed using adrenal histology and hormone testing. RESULTS: Inhalant abuse suppressed appetite and increased energy expenditure. Reduced weight gain and growth were observed in both the toluene and pair-fed groups. Compared to the pair-fed group, and despite normalisation of food intake, the suppression of weight and growth for toluene-exposed rats persisted during abstinence. After exposure, toluene-exposed rats had low fasting blood glucose and insulin compared to the air and pair-fed groups. Consistent with adrenal insufficiency, adrenal hypertrophy and increased basal adrenocorticotropic hormone were observed in the toluene-exposed rats, despite normal basal corticosterone levels. CONCLUSIONS: Inhalant abuse results in negative energy balance, persistent growth impairment, and endocrine changes suggestive of adrenal insufficiency. We conclude that adrenal insufficiency contributes to the negative energy balance phenotype, potentially presenting a significant additional health risk for inhalant users.

Autocrine and Paracrine Regulation of the Murine Skeleton by Osteocyte-Derived Parathyroid Hormone-Related Protein.

Parathyroid hormone-related protein (PTHrP) and parathyroid hormone (PTH) have N-terminal domains that bind a common receptor, PTHR1. N-terminal PTH (teriparatide) and now a modified N-terminal PTHrP (abaloparatide) are US Food and Drug Administration (FDA)-approved therapies for osteoporosis. In physiology, PTHrP does not normally circulate at significant levels, but acts locally, and osteocytes, cells residing within the bone matrix, express both PTHrP and the PTHR1. Because PTHR1 in osteocytes is required for normal bone resorption, we determined how osteocyte-derived PTHrP influences the skeleton. We observed that adult mice with low PTHrP in osteocytes (targeted with the Dmp1(10kb)-Cre) have low trabecular bone volume and osteoblast numbers, but osteoclast numbers were unaffected. In addition, bone size was normal, but cortical bone strength was impaired. Osteocyte-derived PTHrP therefore stimulates bone formation and bone matrix strength, but is not required for normal osteoclastogenesis. PTHrP knockdown and overexpression studies in cultured osteocytes indicate that osteocyte-secreted PTHrP regulates their expression of genes involved in matrix mineralization. We determined that osteocytes secrete full-length PTHrP with no evidence for secretion of lower molecular weight forms containing the N-terminus. We conclude that osteocyte-derived full-length PTHrP acts through both PTHR1 receptor-mediated and receptor-independent actions in a paracrine/autocrine manner to stimulate bone formation and to modify adult cortical bone strength. © 2017 American Society for Bone and Mineral Research.

Increased sphingosine-1-phosphate production in response to osteocyte mechanotransduction.

Over the past few years interest has greatly increased in how the lipid mediator sphingosine-1-phosphate (S1P) influences bone homeostasis. Recent work has postulated multiple effects of S1P on osteoblasts and osteoclasts. Based on these findings, S1P has been proposed as a potential osteoporosis treatment. However, to date, there has been only a single study investigating S1P signalling in the cells that co-ordinate bone metabolism: osteocytes. This study aimed to elucidate the role of S1P signalling in osteocyte mechanotransduction. Utilising 3D cell culture we established the expression profile of all genes related to the S1P signalling system in the Ocy454 osteocyte cell line. Exposure to mechanical loading resulted in a downregulation in Sost, Spns2, the S1P transporter, Sgpl1 and Sgppl1 the enzymes responsible for degradation and dephosphorylation of S1P. These findings, in conjunction with fluid-flow induced upregulation of Sphk1, the kinase responsible for phosphorylation of sphingosine, suggest that mechanical stimulation of osteocytes leads to an increase in intracellular S1P. This was confirmed with mechanical loading of Ocy454 cells rapidly increasing S1P production in conditioned media and protein lysates. These findings strongly suggest an important role for S1P in the response to mechanical loading of bone.

Bone corticalization requires local SOCS3 activity and is promoted by androgen action via interleukin-6.

Long bone strength is determined by its outer shell (cortical bone), which forms by coalescence of thin trabeculae at the metaphysis (corticalization), but the factors that control this process are unknown. Here we show that SOCS3-dependent cytokine expression regulates bone corticalization. Young male and female Dmp1Cre.Socs3 f/f mice, in which SOCS3 has been ablated in osteocytes, have high trabecular bone volume and poorly defined metaphyseal cortices. After puberty, male mice recover, but female corticalization is still impaired, leading to a lasting defect in bone strength. The phenotype depends on sex-steroid hormones: dihydrotestosterone treatment of gonadectomized female Dmp1Cre.Socs3 f/f mice restores normal cortical morphology, whereas in males, estradiol treatment, or IL-6 deletion, recapitulates the female phenotype. This suggests that androgen action promotes metaphyseal corticalization, at least in part, via IL-6 signaling.The strength of long bones is determined by coalescence of trabeculae during corticalization. Here the authors show that this process is regulated by SOCS3 via a mechanism dependent on IL-6 and expression of sex hormones.

High dose dietary vitamin D3 increases bone mass and strength in mice.

Vitamin D plays a critical role in skeletal homeostasis. Vitamin D supplementation is used worldwide to maintain optimal bone health, but the most appropriate level of supplementation remains controversial. This study aimed to determine the effects of varying doses of dietary vitamin D3 on the mechanical properties and morphology of growing bone. Eight-week-old female mice were supplied with one of 3 diets, each containing a different dose of vitamin D3: 1000 IU/kg (control), 8000 IU/kg or 20,000 IU/kg. Mice had ad libitum access to the specialty diet for 4 weeks before they were culled and their tibiae collected for further analysis. The collected tibia underwent three-point bending and reference-point indentation from which their mechanical properties were determined, and cortical and trabecular morphology determined by micro computed tomography. Dietary supplementation with 20,000 IU/kg vitamin D3 resulted in greater ductility (~ 200%) and toughness (~ 150%) compared to the 1000 IU/kg control. The 20,000 IU/kg diet was also associated with significantly greater trabecular bone volume fraction and trabecular number. The 8000 IU/kg diet had no significant effect on trabecular bone mass. We conclude that vitamin D3 supplementation of 20,000 IU/kg during early adulthood leads to tougher bone that is more ductile and less brittle than that of mice supplied with standard levels of dietary vitamin D3 (1000 IU/kg) or 8000 IU/kg. This suggests that dietary vitamin D3 supplementation may increase bone health by improving bone material strength and supports the use of vitamin D3 supplementation, during adolescence, for achieving a higher peak bone mass in adulthood and thereby preventing osteoporosis.

Osteocyte secreted factors inhibit skeletal muscle differentiation.

It is generally accepted that bone and muscle possess the capacity to act in an autocrine, paracrine, or endocrine manner, with a growing body of evidence that suggests muscle can secrete muscle specific cytokines or "myokines", which influence bone metabolism. However, there has been little investigation into the identity of bone specific cytokines that modulate skeletal muscle differentiation and function. This study aimed to elucidate the influence of osteocytes on muscle progenitor cells in vitro and to identify potential bone specific cytokines or "osteokines". We treated C2C12 myoblasts with media collected from differentiated osteocytes (Ocy454 cells) grown in 3D, either under static or fluid flow culture conditions (2 dynes/cm2). C2C12 differentiation was significantly inhibited with a 75% reduction in the number of myofibers formed. mRNA analysis revealed a significant reduction in the expression of myogenic regulatory genes. Cytokine array analysis on the conditioned media demonstrated that osteocytes produce a significant number of cytokines "osteokines" capable of inhibiting myogenesis. Furthermore, we demonstrated that when osteocytes are mechanically activated they induce a greater inhibitory effect on myogenesis compared to a static state. Lastly, we identified the downregulation of numerous cytokines, including Il-6, Il-13, Il-1ß, MIP-1α, and Cxcl9, involved in myogenesis, which may lead to future investigation of the role "osteokines" play in musculoskeletal health and pathology.

The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro.

Although bone responds to its mechanical environment, the cellular and molecular mechanisms underlying the response of the skeleton to mechanical unloading are not completely understood. Osteocytes are the most abundant but least understood cells in bones and are thought to be responsible for sensing stresses and strains in bone. Sclerostin, a product of the SOST gene, is produced postnatally primarily by osteocytes and is a negative regulator of bone formation. Recent studies show that SOST is mechanically regulated at both the mRNA and protein levels. During prolonged bed rest and immobilization, circulating sclerostin increases both in humans and in animal models, and its increase is associated with a decrease in parathyroid hormone. To investigate whether SOST/sclerostin up-regulation in mechanical unloading is a cell-autonomous response or a hormonal response to decreased parathyroid hormone levels, we subjected osteocytes to an in vitro unloading environment achieved by the NASA rotating wall vessel system. To perform these studies, we generated a novel osteocytic cell line (Ocy454) that produces high levels of SOST/sclerostin at early time points and in the absence of differentiation factors. Importantly, these osteocytes recapitulated the in vivo response to mechanical unloading with increased expression of SOST (3.4 ± 1.9-fold, p < 0.001), sclerostin (4.7 ± 0.1-fold, p < 0.001), and the receptor activator of nuclear factor κΒ ligand (RANKL)/osteoprotegerin (OPG) (2.5 ± 0.7-fold, p < 0.001) ratio. These data demonstrate for the first time a cell-autonomous increase in SOST/sclerostin and RANKL/OPG ratio in the setting of unloading. Thus, targeted osteocyte therapies could hold promise as novel osteoporosis and disuse-induced bone loss treatments by directly modulating the mechanosensing cells in bone.

Decline in calcitonin receptor expression in osteocytes with age.

We have previously shown that co-administration of the transient osteoclast inhibitor, salmon calcitonin (sCT), blunts the anabolic effect of parathyroid hormone (PTH) in young rats and increases osteocytic expression of the bone formation inhibitor sclerostin (Sost). To determine whether this also occurs in adult animals, we co-administered sCT with PTH to 6-month-old sham-operated (SHAM) and ovariectomised (OVX) rats. While sCT reduced the stimulatory effect of PTH on serum amino-terminal propeptide of type 1 procollagen levels, in contrast to its influence in young rats, sCT did not reduce the anabolic effect of PTH on femoral bone mineral density, tibial trabecular bone volume or bone formation rate in 6-month-old SHAM or OVX rats. Quantitative real-time PCR analysis of femoral metaphyses collected 1 and 4 h after a single PTH injection confirmed a significant increase in mRNA levels for interleukin 6 (Il6) and ephrinB2 (EfnB2), and a significant reduction in Sost and dentin matrix protein-1 (Dmp1) in response to PTH. However, in contrast to observations in young rats, these effects were not modified by co-administration of sCT, nor did sCT significantly modify Sost, Dmp1, or matrix extracellular phosphoglycoprotein (Mepe) mRNA levels. Furthermore, while CT receptor (CTR) mRNA (Calcr) was readily detected in GFP+ osteocytes isolated from young (3-week-old) DMP1-GFP mice, Calcr levels in osteocytes declined as mice aged, reaching levels that were undetectable in long bone at 49 weeks of age. These data indicate that osteocyte-mediated responses to CT are most likely to be of physiological relevance in young rodents.

Reversing LRP5-dependent osteoporosis and SOST deficiency-induced sclerosing bone disorders by altering WNT signaling activity.

The bone formation inhibitor sclerostin encoded by SOST binds in vitro to low-density lipoprotein receptor-related protein (LRP) 5/6 Wnt co-receptors, thereby inhibiting Wnt/ß-catenin signaling, a central pathway of skeletal homeostasis. Lrp5/LRP5 deficiency results in osteoporosis-pseudoglioma (OPPG), whereas Sost/SOST deficiency induces lifelong bone gain in mice and humans. Here, we analyzed the bone phenotype of mice lacking Sost (Sost(-/-) ), Lrp5 (Lrp5(-/-) ), or both (Sost(-/-) ;Lrp5(-/-) ) to elucidate the mechanism of action of Sost in vivo. Sost deficiency-induced bone gain was significantly blunted in Sost(-/-) ;Lrp5(-/-) mice. Yet the Lrp5 OPPG phenotype was fully rescued in Sost(-/-) ;Lrp5(-/-) mice and most bone parameters were elevated relative to wild-type. To test whether the remaining bone increases in Sost(-/-) ;Lrp5(-/-) animals depend on Lrp6, we treated wild-type, Sost(-/-) , and Sost(-/-) ;Lrp5(-/-) mice with distinct Lrp6 function blocking antibodies. Selective blockage of Wnt1 class-mediated Lrp6 signaling reduced cancellous bone mass and density in wild-type mice. Surprisingly, it reversed the abnormal bone gain in Sost(-/-) and Sost(-/-) ;Lrp5(-/-) mice to wild-type levels irrespective of enhancement or blockage of Wnt3a class-mediated Lrp6 activity. Thus, whereas Sost deficiency-induced bone anabolism partially requires Lrp5, it fully depends on Wnt1 class-induced Lrp6 activity. These findings indicate: first, that OPPG syndrome patients suffering from LRP5 loss-of-function should benefit from principles antagonizing SOST/sclerostin action; and second, that therapeutic WNT signaling inhibitors may stop the debilitating bone overgrowth in sclerosing disorders related to SOST deficiency, such as sclerosteosis, van Buchem disease, and autosomal dominant craniodiaphyseal dysplasia, which are rare disorders without viable treatment options.

Localization of relaxin receptors in arteries and veins, and region-specific increases in compliance and bradykinin-mediated relaxation after in vivo serelaxin treatment.

Relaxin is a potent vasodilator of small resistance arteries and modifies arterial compliance in some systemic vascular beds, yet receptors for relaxin, such as RXFP1, have only been localized to vascular smooth muscle. This study first aimed to localize RXFP1 in rat arteries and veins from different organ beds and determine whether receptors are present in endothelial cells. We then tested the hypothesis that region-specific vascular effects of relaxin may be influenced by the cellular localization of RXFP1 within different blood vessels. The aorta, vena cava, mesenteric artery, and vein had significantly higher (P<0.05) RXFP1 immunostaining in endothelial cells compared with vascular smooth muscle, whereas the femoral artery and vein and small pulmonary arteries had higher (P<0.01) RXFP1 immunostaining in the vascular smooth muscle. Male rats were treated subcutaneously with recombinant human relaxin-2 (serelaxin; 4 µg/h) for 5 d; vasodilation and compliance in mesenteric and femoral arteries and veins were compared with placebo controls. Serelaxin significantly (P=0.04) reduced wall stiffness and increased volume compliance in mesenteric arteries but not in the other vessels examined. This was associated with changes in geometrical properties, and not compositional changes in the extracellular matrix. Serelaxin treatment had no effect on acetylcholine-mediated relaxation but significantly (P<0.001) enhanced bradykinin (BK)-mediated relaxation in mesenteric arteries, involving enhanced nitric oxide but not endothelium-derived hyperpolarization or vasodilatory prostanoids. In conclusion, there is differential distribution of RXFP1 on endothelial and smooth muscle across the vasculature. In rats, mesenteric arteries exhibit the greatest functional response to chronic serelaxin treatment.

Impaired effect of relaxin on vasoconstrictor reactivity in spontaneous hypertensive rats.

Relaxin is thought to be involved in vasodilation to pregnancy by increasing endothelium-dependent vasodilation and compliance, and decreasing myogenic reactivity. Primary (essential) hypertension predisposes to circulatory maladaptation and subsequent gestational hypertensive disease. This study aimed to determine that vascular responses to chronic exposure to relaxin are impaired in young female rats with primary hypertension. In 10-12 weeks old Wistar-Hannover rats (WHR) and spontaneous hypertensive rats (SHR), we determined vascular responses in isolated kidney and mesenteric arteries after 5-days of chronic exposure to relaxin (4 µg/h) or placebo. SHR show decreased sensitivity to phenylephrine (by 67%, p<0.01) and renal perfusion flow (RPFF, by 19%, p<0.01), but no changes in flow-mediated vasodilation, myogenic reactivity or vascular compliance. In WHR, relaxin stimulated flow-mediated vasodilation (2.67 fold, from 48 ± 9 to 18 ± 4 µl/min, p = 0.001), inhibited myogenic reactivity (from -1 ± 2 to 7 ± 3 µm/10 mmHg, p = 0.01), and decreased sensitivity to phenylephrine (28%, from 1.39 ± 0.08 to 1.78 ± 0.10 µM, p<0.01), but left compliance and RPFF unchanged. NO-blockade by L-NAME diminished most relaxin-mediated responses. In SHR, the vasodilator effects of relaxin were blunted for myogenic reactivity and sensitivity to phenylephrine, with similar effects on flow-mediated vasodilation, compliance, RPFF and equal Rxfp1 (relaxin family peptide receptor) gene expression, as compared to WHR. Primary hypertension blunts both the relaxin-induced inhibition of myogenic reactivity and α-adrenergic vasoconstrictor response, independent from Rxfp1 gene expression, while the relaxin-dependent enhanced flow-mediated vasodilation remains intact. This implies selective resistance to relaxin in young subjects suffering from primary hypertension.