Thermoneutral Housing has Limited Effects on Social Isolation-Induced Bone Loss in Male C57BL/6J Mice.

Social isolation stress has numerous known negative health effects, including increased risk for cardiovascular disease, dementia, as well as overall mortality. The impacts of social isolation on skeletal health, however, have not been thoroughly investigated. We previously found that four weeks of social isolation through single housing led to a significant reduction in trabecular and cortical bone in male, but not female, mice. One possible explanation for these changes in male mice is thermal stress due to sub-thermoneutral housing. Single housing at room temperature (~20-25°C)-below the thermoneutral range of mice (~26-34°C)-may lead to cold stress, which has known negative effects on bone. Therefore, the aim of this study was to test the hypothesis that housing mice near thermoneutrality, thereby ameliorating cold-stress, will prevent social isolation-induced bone loss in male C57BL/6J mice. 16-week-old mice were randomized into social isolation (1 mouse/cage) or grouped housing (4 mice/cage) at either room temperature (~23°C) or in a warm temperature incubator (~28°C) for four weeks (N=8/group). As seen in our previous studies, isolated mice at room temperature had significantly reduced bone parameters, including femoral bone volume fraction (BV/TV), bone mineral density (BMD), and cortical thickness. Contrary to our hypothesis, these negative effects on bone were not ameliorated by thermoneutral housing. Social isolation increased glucocorticoid-related gene expression in bone and Ucp1 and Pdk4 expression in BAT across temperatures, while thermoneutral housing increased percent lipid area and decreased Ucp1 and Pdk4 expression in BAT across housing conditions. Overall, our data suggest social isolation-induced bone loss is not a result of thermal stress from single housing and provides a key insight into the mechanism mediating the effects of isolation on skeletal health.

Calorie restriction in mice impairs cortical but not trabecular peak bone mass by suppressing bone remodeling.

Calorie restriction (CR) can lead to weight loss and decreased substrate availability for bone cells. Ultimately, this can lead to impaired peak bone acquisition in children and adolescence and bone loss in adults. But the mechanisms that drive diet-induced bone loss in humans are not well characterized. To explore those in greater detail, we examined the impact of 30% CR for 4 and 8 wk in both male and female 8-wk-old C57BL/6 J mice. Body composition, areal bone mineral density (aBMD), skeletal microarchitecture by micro-CT, histomorphometric parameters, and in vitro trajectories of osteoblast and adipocyte differentiation were examined. After 8 wk, CR mice lost weight and exhibited lower femoral and whole-body aBMD vs ad libitum (AL) mice. By micro-CT, CR mice had lower cortical bone area fraction vs AL mice, but males had preserved trabecular bone parameters and females showed increased bone volume fraction compared to AL mice. Histomorphometric analysis revealed that CR mice had a profound suppression in trabecular as well as endocortical and periosteal bone formation in addition to reduced bone resorption compared to AL mice. Bone marrow adipose tissue was significantly increased in CR mice. In vitro, the pace of adipogenesis in bone marrow stem cells was greatly accelerated with higher markers of adipocyte differentiation and more oil red O staining, whereas osteogenic differentiation was reduced. qRT-PCR and western blotting suggested that the expression of Wnt16 and the canonical ß-catenin pathway was compromised during CR. In sum, CR causes impaired peak cortical bone mass due to a profound suppression in bone remodeling. The increase in marrow adipocytes in vitro and in vivo is related to both progenitor recruitment and adipogenesis in the face of nutrient insufficiency. Long-term CR may lead to lower bone mass principally in the cortical envelope, possibly due to impaired Wnt signaling.

Calorie restriction led to impaired bone mass and increased accumulation of bone marrow adipose tissue. During the development of bone-fat imbalance due to calorie restriction, bone remodeling was notably inhibited. Calorie restriction may shift the differentiation of bone marrow stem cells toward adipocytes instead of osteoblasts. This process involves a disruption in the canonical Wnt signaling pathway.

Social and maternal behavior in mesoderm specific transcript (Mest)-deficient mice.

Mesoderm specific transcript (Mest)/paternally expressed gene-1 (Peg1) is an imprinted gene expressed predominantly from the paternal allele. Aberrations in maternal behavior were previously reported in a Mest global knockout mouse (Mesttm1Masu). In this study, we performed in-depth social and maternal behavioral testing in a mouse model of Mest inactivation developed in our laboratory (Mesttm1.2Rkz). Mice with paternal allele inactivation (MestpKO) did not show anxiety after testing in the elevated plus maze, open field trial, and marble burying; nor depression-like behaviors in the tail suspension test. MestpKO showed normal social behaviors and memory/cognition in the three-chamber box test and the novel object recognition test, respectively. Primiparous MestpKO and MestgKO (biallelic Mest inactivation) female mice exhibited normal nest building and maternal behavior; and, virgin MestpKO and MestgKO female mice showed normal maternal instinct. Analyses of gene expression in adult hypothalamus, embryonic day 14.5 whole brain and adult whole brain demonstrated full abrogation of Mest mRNA in MestpKO and MestgKO mice with no effect on miR-335 expression. Our data indicates no discernible impairments in object recognition memory, social behavior or maternal behavior resulting from loss of Mest. The basis for the differences in maternal phenotypic behaviors between Mesttm1Masu and Mesttm1.2Rkz is not known.

Deletion of α-Synuclein in Prrx1-positive cells causes partial loss of function in the central nervous system (CNS) but does not affect ovariectomy induced bone loss.

α-Synuclein is a small 140 amino acid polypeptide encoded by the Snca gene that is highly expressed in neural tissue, but it is also found in osteoblasts, erythroblasts, macrophages, and adipose tissue. Previously, using co-expression network analysis we found that Snca was a key regulator of skeletal homeostasis, and its deletion partially prevented bone loss after ovariectomy (OVX). Here we tested the hypothesis that Snca deletion in mesenchymal progenitors using the Prrx1Cre (Prrx1, Paired-related homeobox 1) limb enhancer would protect bone mass after OVX. Prrx1Cre;Sncafl/fl and littermate controls (Sncafl/fl) were sham operated or ovariectomized (OVX) at 8 weeks of age and sacrificed at 20 weeks. Independently, eight-week female and male Prrx1Cre;Sncafl/fl mice and littermate controls were administered a high fat (60% fat) or low fat (10% fat) diet for 15 weeks. Bone loss was not prevented in either genotype after ovariectomy, but the Prrx1Cre;Sncafl/fl. mice were partially protected from weight gain after OVX and high fat diet (HFD). Serum catecholamine levels were lower in the Prrx1Cre;Sncafl/fl both on a low fat diet (LFD) and HFD versus fl/fl controls. Importantly, mutant mice exhibited a number of physical and behavioral phenotypes that were associated with conditional deletion of Snca in several brain regions. Cells labeled with Prrx1 were noted throughout the central nervous system (CNS). These data support earlier preliminary reports of Prrx1 expression in neural progenitors, and raise a cautionary note about the evaluation of skeletal and body composition phenotypes when using this Cre driver to study osteoprogenitor development.

Thyroid hormone influences brain gene expression programs and behaviors in later generations by altering germ line epigenetic information.

Genetic factors do not fully account for the relatively high heritability of neurodevelopmental conditions, suggesting that non-genetic heritable factors contribute to their etiology. To evaluate the potential contribution of aberrant thyroid hormone status to the epigenetic inheritance of neurological phenotypes, we examined genetically normal F2 generation descendants of mice that were developmentally overexposed to thyroid hormone due to a Dio3 mutation. Hypothalamic gene expression profiling in postnatal day 15 F2 descendants on the paternal lineage of ancestral male and female T3-overexposed mice revealed, respectively, 1089 and 1549 differentially expressed genes. A large number of them, 675 genes, were common to both sets, suggesting comparable epigenetic effects of thyroid hormone on both the male and female ancestral germ lines. Oligodendrocyte- and neuron-specific genes were strongly overrepresented among genes showing, respectively, increased and decreased expression. Altered gene expression extended to other brain regions and was associated in adulthood with decreased anxiety-like behavior, increased marble burying and reduced physical activity. The sperm of T3-overexposed male ancestors revealed significant hypomethylation of CpG islands associated with the promoters of genes involved in the early development of the central nervous system. Some of them were candidates for neurodevelopmental disorders in humans including Nrg3, Nrxn1, Gabrb3, Gabra5, Apba2, Grik3, Reln, Nsd1, Pcdh8, En1, and Elavl2. Thus, developmental levels of thyroid hormone influence the epigenetic information of the germ line, disproportionately affecting genes with critical roles in early brain development, and leading in future generations to disease-relevant alterations in postnatal brain gene expression and adult behavior.

Adult onset of type 3 deiodinase deficiency in mice alters brain gene expression and increases locomotor activity.

Constitutive loss of the type 3 deiodinase (DIO3) causes abnormally increased levels of thyroid hormone action in the developing and adult brain, leading to an array of behavioral abnormalities. To determine to what extent those phenotypes derive from a lack of DIO3 in the adult brain, versus developmental consequences, we created a mouse model of conditional DIO3 inactivation. Mice carrying "floxed" Dio3 alleles and a tamoxifen-inducible cre transgene were injected with tamoxifen at two months of age. Compared to oil-injected controls, the brain tissue of these mice showed a 75-80% decrease in DIO3 activity and 85-95% Dio3 mRNA was expressed from recombinant alleles. Mice with adult DIO3 deficiency did not show significant differences in growth, serum thyroid hormone parameters or behaviors related to anxiety and depression. However, female mice exhibited elevated locomotor activity and increased marble-burying behavior. They also manifested relatively modest alterations in the expression of T3-dependent genes and genes related to hyperactivity in a sex- and region-specific manner. Upon thyroid hormone treatment, the expression response of T3-regulated genes was generally more pronounced in DIO3-deficient female mice than in female controls, while the opposite effect of altered genotype was noticed in males. The extent of the molecular and behavioral phenotypes of adult-onset DIO3 deficiency suggests that a substantial proportion of the neurological abnormalities caused by constitutive DIO3 deficiency has a developmental origin. However, our results show that DIO3 in the adult brain also influences behavior and sensitivity to thyroid hormone action in a sexually dimorphic fashion.

The Type 3 Deiodinase: Epigenetic Control of Brain Thyroid Hormone Action and Neurological Function.

Thyroid hormones (THs) influence multiple processes in the developing and adult central nervous system, and their local availability needs to be maintained at levels that are tailored to the requirements of their biological targets. The local complement of TH transporters, deiodinase enzymes, and receptors is critical to ensure specific levels of TH action in neural cells. The type 3 iodothyronine deiodinase (DIO3) inactivates THs and is highly present in the developing and adult brain, where it limits their availability and action. DIO3 deficiency in mice results in a host of neurodevelopmental and behavioral abnormalities, demonstrating the deleterious effects of TH excess, and revealing the critical role of DIO3 in the regulation of TH action in the brain. The fact the Dio3 is an imprinted gene and that its allelic expression pattern varies across brain regions and during development introduces an additional level of control to deliver specific levels of hormone action in the central nervous system (CNS). The sensitive epigenetic nature of the mechanisms controlling the genomic imprinting of Dio3 renders brain TH action particularly susceptible to disruption due to exogenous treatments and environmental exposures, with potential implications for the etiology of human neurodevelopmental disorders.

Inhibition of osteoclast differentiation and collagen antibody-induced arthritis by CTHRC1.

Collagen triple helix repeat-containing1 (Cthrc1) has previously been implicated in osteogenic differentiation and positive regulation of bone mass, however, the underlying mechanisms remain unclear. Here we characterized the bone phenotype of a novel Cthrc1 null mouse strain using bone histomorphometry, µCT analysis and functional readouts for bone strength. In male Cthrc1 null mice both trabecular bone as well as cortical bone formation was impaired, whereas in female Cthrc1 null mice only trabecular bone parameters were altered. Novel and highly specific monoclonal antibodies revealed that CTHRC1 is expressed by osteocytes and osteoblasts, but not osteoclasts. Furthermore, Cthrc1 null mice exhibited increased bone resorption with increased number of osteoclast and increased osteoclast activity together with enhanced expression of osteoclastogenic genes such as c-Fos, Rankl, Trap, and Nfatc1. Differentiation of bone marrow-derived monocytes isolated from Cthrc1 null mice differentiated into osteoclasts as effectively as those from wildtype mice. In the presence of CTHRC1 osteoclastogenic differentiation of bone marrow-derived monocytes was dramatically inhibited as was functional bone resorption by osteoclasts. This process was accompanied by downregulation of osteoclastogenic marker genes, indicating that extrinsically derived CTHRC1 is required for such activity. In vitro, CTHRC1 had no effect on osteogenic differentiation of bone marrow stromal cells, however, calvarial osteoblasts from Cthrc1 null mice exhibited reduced osteogenic differentiation compared to osteoblasts from wildtypes. In a collagen antibody-induced arthritis model Cthrc1 null mice suffered significantly more severe inflammation and joint destruction than wildtypes, suggesting that CTHRC1 expressed by the activated synoviocytes has anti-inflammatory effects. Mechanistically, we found that CTHRC1 inhibited NFκB activation by preventing IκBα degradation while also inhibiting ERK1/2 activation. Collectively our studies demonstrate that CTHRC1 secreted from osteocytes and osteoblasts functions as an inhibitor of osteoclast differentiation via inhibition of NFκB-dependent signaling. Furthermore, our data suggest that CTHRC1 has potent anti-inflammatory properties that limit arthritic joint destruction.

Decreased anxiety- and depression-like behaviors and hyperactivity in a type 3 deiodinase-deficient mouse showing brain thyrotoxicosis and peripheral hypothyroidism.

Hypo- and hyperthyroid states, as well as functional abnormalities in the hypothalamic-pituitary-thyroid axis have been associated with psychiatric conditions like anxiety and depression. However, the nature of this relationship is poorly understood since it is difficult to ascertain the thyroid status of the brain in humans. Data from animal models indicate that the brain exhibits efficient homeostatic mechanisms that maintain local levels of the active thyroid hormone, triiodothyronine (T3) within a narrow range. To better understand the consequences of peripheral and central thyroid status for mood-related behaviors, we used a mouse model of type 3 deiodinase (DIO3) deficiency (Dio3 -/- mouse). This enzyme inactivates thyroid hormone and is highly expressed in the adult central nervous system. Adult Dio3 -/- mice exhibit elevated levels of T3-dependent gene expression in the brain, despite peripheral hypothyroidism as indicated by low circulating levels of thyroxine and T3. Dio3 -/- mice of both sexes exhibit hyperactivity and significantly decreased anxiety-like behavior, as measured by longer time spent in the open arms of the elevated plus maze and in the light area of the light/dark box. During the tail suspension, they stayed immobile for a significantly shorter time than their wild-type littermates, suggesting decreased depression-like behavior. These results indicate that increased thyroid hormone in the brain, not necessarily in peripheral tissues, correlates with hyperactivity and with decreases in anxiety and depression-like behaviors. Our results also underscore the importance of DIO3 as a determinant of behavior by locally regulating the brain levels of thyroid hormone.

MCT8 Deficiency in Male Mice Mitigates the Phenotypic Abnormalities Associated With the Absence of a Functional Type 3 Deiodinase.

Mice deficient in the type 3 deiodinase (D3KO mice) manifest impaired clearance of thyroid hormone (TH), leading to elevated levels of TH action during development. This alteration causes reduced neonatal viability, growth retardation, and central hypothyroidism. Here we examined how these phenotypes are affected by a deficiency in the monocarboxylate transporter 8 (MCT8), which is a major contributor to the transport of the active thyroid hormone, T3, into the cell. MCT8 deficiency eliminated the neonatal lethality of type 3 deiodinase (D3)-deficient mice and significantly ameliorated their growth retardation. Double-mutant newborn mice exhibited similar peripheral thyrotoxicosis and increased brain expression of T3-dependent genes as mice with D3 deficiency only. Later in neonatal life and adulthood, double-mutant mice manifested central and peripheral TH status similar to mice with single MCT8 deficiency, with low serum T4, elevated serum TSH and T3, and decreased T3-dependent gene expression in the hypothalamus. In double-mutant adult mice, both thyroid gland size and the hypothyroidism-induced rise in TSH were greater than those in mice with single D3 deficiency but less than those in mice with MCT8 deficiency alone. Our results demonstrate that the marked phenotypic abnormalities observed in the D3-deficient mouse, including perinatal mortality, growth retardation, and central hypothyroidism in adult animals, require expression of MCT8, confirming the interdependent relationship between the TH transport into cells and the deiodination processes.

The Type 3 Deiodinase Is a Critical Determinant of Appropriate Thyroid Hormone Action in the Developing Testis.

Timely and appropriate levels of thyroid hormone (TH) signaling are necessary to ensure normal developmental outcomes in many tissues. Studies using pharmacological models of altered TH status have revealed an influence of these hormones on testis development and size, but little is known about the role of endogenous determinants of TH action in the developing male gonads. Using a genetic approach, we demonstrate that the type 3 deiodinase (D3), which inactivates TH and protects developing tissues from undue TH action, is a key factor. D3 is highly expressed in the developing testis, and D3-deficient (D3KO) mice exhibit thyrotoxicosis and cell proliferation arrest in the neonatal testis, resulting in an approximately 75% reduction in testis size. This is accompanied by larger seminiferous tubules, impaired spermatogenesis, and a hormonal profile indicative of primary hypogonadism. A deficiency in the TH receptor-α fully normalizes testis size and adult testis gene expression in D3KO mice, indicating that the effects of D3 deficiency are mediated through this type of receptor. Similarly, genetic deficiencies in the D2 or in the monocarboxylate transporter 8 partially rescue the abnormalities in testis size and gonadal axis gene expression featured in the D3KO mice. Our study highlights the testis as an important tissue in which determinants of TH action coordinately converge to ensure normal development and identifies D3 as a critical factor in testis development and in testicular protection from thyrotoxicosis.

Cthrc1 controls adipose tissue formation, body composition, and physical activity.

OBJECTIVE: This study investigated the effects of loss of Cthrc1 on adipogenesis, body composition, metabolism, physical activity, and muscle physiology. METHODS: Complete metabolic and activity monitoring as well as grip strength measurements and muscle myography was performed in Cthrc1 null and wildtype mice. RESULTS: Compared to wildtypes, Cthrc1 null mice had similar body weights but significantly reduced energy expenditure, decreased lean mass, and increased fat mass, especially visceral fat. In vitro studies demonstrated that Cthrc1 inhibited adipocyte differentiation as well as PPAR and CREB reporter activity, while preadipocytes isolated from Cthrc1 null mice exhibited enhanced adipogenic differentiation. Voluntary physical activity in Cthrc1 null mice as assessed by wheel running was reduced to approximately half the distance covered by wildtypes. Reduced grip strength was observed in Cthrc1 null mice at the age of 15 weeks or older with reduced performance and mass of hyphenate muscle. In the brain, Cthrc1 expression was most prominent in neurons of thalamic and hypothalamic nuclei with evidence for secretion into the circulation in the median eminence. CONCLUSIONS: Our data indicate that Cthrc1 regulates body composition through inhibition of adipogenesis. In addition, central Cthrc1 may be a mediator of muscle function and physical activity.

Elevated plasma levels of the pituitary hormone Cthrc1 in individuals with red hair but not in patients with solid tumors.

BACKGROUND: An increasing number of studies report that Cthrc1 is expressed in various cancer cells. The present study sought to identify which cells in tumors and remodeling tissues express Cthrc1 and investigate the range of circulating human Cthrc1 levels in health and disease. METHODOLOGY/PRINCIPLE FINDINGS: Highly specific monoclonal antibodies were generated to detect Cthrc1 by ELISA in plasma and in tissues by immunohistochemistry. In human colon, gastric, breast, endometrial, pancreatic, kidney, lung and skin cancer, Cthrc1 was expressed by activated stromal cells and not the cancer cells themselves. Similarly, conditions evoking tissue remodeling, such as wound repair or angiotensin II-mediated hypertension, induced Cthrc1 expression in interstitial and adventitial fibroblasts and perivascular stromal cells. Levels of Cthrc1 in plasma from healthy subjects were near the lower detection limit except for individuals with red hair, who had up to several hundred fold higher levels. Elevated Cthrc1 was also found in patients with diabetes, inflammatory conditions, and infections, but not solid tumors. Transgenic mouse studies suggested that Cthrc1 expression by stromal cells does not contribute to circulating levels. In human pituitaries, Cthrc1 was expressed in the anterior and intermediate lobes with unencapsulated Cthrc1 accumulations typically surrounded by chromophobe cells. CONCLUSIONS: We identify Cthrc1 as a marker for activated stromal cells. Cthrc1 is a pituitary hormone with significantly elevated levels in subjects carrying variant alleles of the melanocortin-1 receptor as wells as in patients with inflammatory conditions.

Cthrc1, a novel circulating hormone regulating metabolism.

BACKGROUND: We discovered the gene Collagen Triple Helix Repeat Containing 1 (Cthrc1) and reported its developmental expression and induction in adventitial cells of injured arteries and dermal cells of skin wounds. The role of Cthrc1 in normal adult tissues has not yet been determined. METHODOLOGY/PRINCIPAL FINDINGS: We generated mutant mice with a novel Cthrc1 null allele by homologues recombination. Cthrc1 null mice appeared developmentally normal. On the C57BL/6J background, livers from Cthrc1 null mice accumulated vast quantities of lipid, leading to extensive macrovesicular steatosis. Glycogen levels in skeletal muscle and liver of Cthrc1 null mice on the 129S6/SvEv background were significantly increased. However, Cthrc1 expression is not detectable in these tissues in wild-type mice, suggesting that the lipid and glycogen storage phenotype may be a secondary effect due to loss of Cthrc1 production at a distant site. To investigate potential hormonal functions of Cthrc1, tissues from adult mice and pigs were examined for Cthrc1 expression by immunohistochemistry with monoclonal anti-Cthrc1 antibodies. In pigs, Cthrc1 was detected around chromophobe cells of the anterior pituitary, and storage of Cthrc1 was observed in colloid-filled follicles and the pituitary cleft. Pituitary follicles have been observed in numerous vertebrates including humans but none of the known pituitary hormones have hitherto been detected in them. In C57BL/6J mice, however, Cthrc1 was predominantly expressed in the paraventricular and supraoptic nucleus of the hypothalamus but not in the posterior pituitary. In human plasma, we detected Cthrc1 in pg/ml quantities and studies with (125)I-labeled Cthrc1 revealed a half-life of 2.5 hours in circulation. The highest level of Cthrc1 binding was observed in the liver. CONCLUSIONS: Cthrc1 has characteristics of a circulating hormone generated from the anterior pituitary, hypothalamus and bone. Hormonal functions of Cthrc1 include regulation of lipid storage and cellular glycogen levels with potentially broad implications for cell metabolism and physiology.

Characterization of Pdgfrb-Cre transgenic mice reveals reduction of ROSA26 reporter activity in remodeling arteries.

With the intention to modulate gene expression in vascular mural cells of remodeling vessels, we generated and characterized transgenic mouse lines with Cre recombinase under the control of the platelet-derived growth factor receptor-ß promoter, referred to as Tg(Pdgfrb-Cre)(35Vli) . Transgenic mice were crossed with the Gt(ROSA)26Sor(tm1Sor) strain and examined for Cre activation by ß-galactosidase activity, which was compared with endogenous Pdgfrb expression. In addition, Pdgfrb-Cre mice were used to drive expression of a conditional myc-tagged Cthrc1 transgene. There was good overlap of ß-galactosidase activity with endogenous Pdgfrb immunoreactivity. However, dedifferentiation of vascular mural cells induced by carotid artery ligation revealed a dramatic discrepancy between ROSA26 reporter activity and Pdgfrb promoter driven Cre dependent myc-tagged Cthrc1 transgene expression. Our studies demonstrate the capability of the Pdgfrb-Cre mouse to drive conditional transgene expression as a result of prior Cre-mediated recombination in tissues known to express endogenous Pdgfrb. In addition, the study shows that ROSA26 promoter driven reporter mice are not suitable for lineage marking of smooth muscle in remodeling blood vessels.

Mitochondrial signal peptidases of yeast: the rhomboid peptidase Pcp1 and its substrate cytochrome C peroxidase.

The rhomboid peptidase Pcp1 of yeast is the first mitochondrial enzyme of this new class of serine peptidases. Pcp1 is an integral part of the inner membrane and was identified by its signal peptidase activity responsible for processing of the intermediate of cytochrome c peroxidase (iCcp1) to the mature enzyme. Here we describe studies on the expression of the PCP1 gene. Proteolytic processing of Pcp1 itself was found. The precursor and the intermediate of Ccp1 were localized to the inner membrane. The results confirm our previous report on a two-step processing pathway of cytochrome c peroxidase and the identification of the signal peptidases involved.