Luciane P. Capelo

The Thyroid Hormone Receptor β‐Specific Agonist GC‐1 Selectively Affects the Bone Development of Hypothyroid Rats

We investigated the effects of GC‐1, a TRβ‐selective thyromimetic, on bone development of hypothyroid rats. Whereas T3 reverted the IGF‐I deficiency and the skeletal defects caused by hypothyroidism, GC‐1 had no effect on serum IGF‐I or on IGF‐I protein expression in the epiphyseal growth plate of the femur, but induced selective effects on bone development. Our findings indicate that T3 exerts some essential effects on bone development that are mediated by TRβ1.

β1 Adrenergic receptor is key to cold- and diet-induced thermogenesis in mice

Brown adipose tissue (BAT) is predominantly regulated by the sympathetic nervous system (SNS) and the adrenergic receptor signaling pathway. Knowing that a mouse with triple β-receptor knockout (KO) is cold intolerant and obese, we evaluated the independent role played by the β(1) isoform in energy homeostasis. First, the 30  min i.v. infusion of norepinephrine (NE) or the β(1) selective agonist dobutamine (DB) resulted in similar interscapular BAT (iBAT) thermal response in WT mice. Secondly, mice with targeted disruption of the β(1) gene (KO of β(1) adrenergic receptor (β(1)KO)) developed hypothermia during cold exposure and exhibited decreased iBAT thermal response to NE or DB infusion. Thirdly, when placed on a high-fat diet (HFD; 40% fat) for 5 weeks, β(1)KO mice were more susceptible to obesity than WT controls and failed to develop diet-induced thermogenesis as assessed by BAT Ucp1 mRNA levels and oxygen consumption. Furthermore, β(1)KO mice exhibited fasting hyperglycemia and more intense glucose intolerance, hypercholesterolemia, and hypertriglyceridemia when placed on the HFD, developing marked non-alcoholic steatohepatitis. In conclusion, the β(1) signaling pathway mediates most of the SNS stimulation of adaptive thermogenesis.

Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton.

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To study this, pregnant mice were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved up to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocytes, in addition to a slight delay in endochondral and intramembranous ossification. This suggests that up to E16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly ( approximately 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast, D2 mRNA expression increased significantly by E18.5 and markedly ( approximately 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development.

The monocarboxylate transporter 8 and L-type amino acid transporters 1 and 2 are expressed in mouse skeletons and in osteoblastic MC3T3-E1 cells.

BACKGROUND Several plasma membrane transporters have been shown to mediate the cellular influx and/or efflux of iodothyronines, including the sodium-independent organic anion co-transporting polypeptide 1 (OATP1), the sodium taurocholate co-transporting polypeptide (NTCP), the L-type amino acid transporter 1 (LAT1) and 2 (LAT2), and the monocarboxylate transporter 8 (MCT8). The aim of this study was to investigate if the mRNAs of these transporters were expressed and regulated by thyroid hormone (TH) in mouse calvaria-derived osteoblastic MC3T3-E1 cells and in the fetal and postnatal bones of mice. METHODS The mRNA expression of the iodothyronine transporters was investigated with real-time polymerase chain reaction analysis in euthyroid and hypothyroid fetuses and litters of mice and in MC3T3-E1 cells treated with increasing doses of triiodothyronine (T(3); 10(-10) to 10(-6) M) or with 10(-8) M T(3) for 1-9 days. RESULTS MCT8, LAT1, and LAT2 mRNAs were detected in fetal and postnatal femurs and in MC3T3-E1 cells, while OATP1 and NTCP mRNAs were not. LAT1 and LAT2 mRNAs were not affected by TH status in vivo or in vitro or by the stage of bone development or osteoblast maturation (analyzed by the expression of osteocalcin and alkaline phosphatase, which are key markers of osteoblastic differentiation). In contrast, the femoral mRNA expression of MCT8 decreased significantly during post-natal development, whereas MCT8 mRNA expression increased as MC3T3-E1 cells differentiated. We also showed that MCT8 mRNA was up-regulated in the femur of hypothyroid animals, and that it was down-regulated by treatment with T(3) in MC3T3-E1 cells. CONCLUSIONS This is the first study to demonstrate the mRNA expression of LAT1, LAT2, and MCT8 in the bone tissue of mice and in osteoblast-like cells. In addition, the pattern of MCT8 expression observed in vivo and in vitro suggests that MCT8 may be important to modulate TH effects on osteoblast differentiation and on bone development and metabolism.

Double disruption of α2A- and α2C -adrenoceptors results in sympathetic hyperactivity and high-bone-mass phenotype

Evidence demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via β2‐adrenoceptor (β2‐AR) signaling. Here we show that female mice with chronic sympathetic hyperactivity owing to double knockout of adrenoceptors that negatively regulate norepinephrine release, α2A‐AR and α2C‐AR (α2A/α2C‐ARKO), present an unexpected and generalized phenotype of high bone mass with decreased bone resorption and increased formation. In α2A/α2C‐ARKO versus wild‐type (WT) mice, micro–computed tomographic (µCT) analysis showed increased, better connected, and more plate‐shaped trabeculae in the femur and vertebra and increased cortical thickness in the vertebra, whereas biomechanical analysis showed increased tibial and femoral strength. Tibial mRNA expression of tartrate‐resistant acid phosphatase (TRACP) and receptor activator of NF‐κB (RANK), which are osteoclast‐related factors, was lower in knockout (KO) mice. Plasma leptin and brain mRNA levels of cocaine amphetamine–regulated transcript (CART), which are factors that centrally affect bone turnover, and serum levels of estradiol were similar between mice strains. Tibial β2‐AR mRNA expression also was similar in KO and WT littermates, whereas α2A‐, α2B‐ and α2C‐AR mRNAs were detected in the tibia of WT mice and in osteoblast‐like MC3T3‐E1 cells. By immunohistochemistry, we detected α2A‐, α2B‐, α2C‐ and β2‐ARs in osteoblasts, osteoclasts, and chondrocytes of 18.5‐day‐old mouse fetuses and 35‐day‐old mice. Finally, we showed that isolated osteoclasts in culture are responsive to the selective α2‐AR agonist clonidine and to the nonspecific α‐AR antagonist phentolamine. These findings suggest that β2‐AR is not the single adrenoceptor involved in bone turnover regulation and show that α2‐AR signaling also may mediate the SNS actions in the skeleton.

The Thyroid Hormone Receptor (TR) β-Selective Agonist GC-1 Inhibits Proliferation But Induces Differentiation and TR β mRNA Expression in Mouse and Rat Osteoblast-Like Cells

Previous studies showed anabolic effects of GC-1, a triiodothyronine (T3) analogue that is selective for both binding and activation functions of thyroid hormone receptor (TR) β1 over TRα1, on bone tissue in vivo. The aim of this study was to investigate the responsiveness of rat (ROS17/2.8) and mouse (MC3T3-E1) osteoblast-like cells to GC-1. As expected, T3 inhibited cellular proliferation and stimulated mRNA expression of osteocalcin or alkaline phosphatase in both cell lineages. Whereas equimolar doses of T3 and GC-1 equally affected these parameters in ROS17/2.8 cells, the effects of GC-1 were more modest compared to those of T3 in MC3T3-E1 cells. Interestingly, we showed that there is higher expression of TRα1 than TRβ1 mRNA in rat (~20–90%) and mouse (~90–98%) cell lineages and that this difference is even higher in mouse cells, which highlights the importance of TRα1 to bone physiology and may partially explain the modest effects of GC-1 in comparison with T3 in MC3T3-E1 cells. Nevertheless, we showed that TRβ1 mRNA expression increases (~2.8- to 4.3-fold) as osteoblastic cells undergo maturation, suggesting a key role of TRβ1 in mediating T3 effects in the bone forming cells, especially in mature osteoblasts. It is noteworthy that T3 and GC-1 induced TRβ1 mRNA expression to a similar extent in both cell lineages (~2- to 4-fold), indicating that both ligands may modulate the responsiveness of osteoblasts to T3. Taken together, these data show that TRβ selective T3 analogues have the potential to directly induce the differentiation and activity of osteoblasts.

Hormone-regulated expression and distribution of versican in mouse uterine tissues

BackgroundRemodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination.MethodsUteri from mice in all phases of the estrous cycle, and animals subjected to ovariectomy and hormone replacement were collected for immunoperoxidase staining for versican, as well as PCR and quantitative Real Time PCR.ResultsIn diestrus and proestrus, VER was exclusively expressed in the endometrial stroma. In estrus and metaestrus, VER was present in both endometrial stroma and myometrium. In OVX mice, VER immunoreaction was abolished in all uterine tissues. VER expression was restored by E2, MPA and E2+MPA treatments. Real Time PCR analysis showed that VER expression increases considerably in the MPA-treated group. Analysis of mRNA identified isoforms V0, V1 and V3 in the mouse uterus.ConclusionThese results show that the expression of versican in uterine tissues is modulated by ovarian steroid hormones, in a tissue-specific manner. VER is induced in the myometrium exclusively by E2, whereas MPA induces VER deposition only in the endometrial stroma.

BMPR-II is dispensable for formation of the limb skeleton

Initiation of BMP signaling is dependent upon activation of Type I BMP receptor by constitutively active Type II BMP receptor. Three Type II BMP receptors have been identified; Acvr2a and Acvr2b serve as receptors for BMPs and for activin‐like ligands whereas BMPR‐II functions only as a BMP receptor. As BMP signaling is required for endochondral ossification and loss of either Acvr2a or Acvr2b is not associated with deficits in limb development, we hypothesized that BMPR‐II would be essential for BMP signaling during skeletogenesis. We removed BMPR‐II from early limb mesoderm by crossing BMPR‐II floxed mice with those carrying the Prx1‐Cre transgene. Mice lacking limb expression of BMPR‐II have normal skeletons that could not be distinguished from control littermates. From these data, we conclude that BMPR‐II is not required for endochondral ossification in the limb where loss of BMPR‐II may be compensated by BMP utilization of Acvr2a and Acvr2b. genesis, 00:1‐6, 2011.

Gene signature of the embryonic meniscus

The meniscus is a fibrocartilagenous disc in the knee that protects the joint from damage. Meniscal injuries are common, however repair efforts are largely unsuccessful and are not able to prevent the degenerative changes that result in development of osteoarthritis. Tissue regeneration in adults often recapitulates events of embryonic development, suggesting the regulatory pathways controlling morphogenesis are candidate repair signals. Here we use laser capture microdissection to collect mouse embryonic day 16 (E16) meniscus, articular cartilage, and cruciate ligaments. RNA isolated from these tissues was then used to perform genome‐wide microarray analysis. We found 38 genes were differentially expressed between E16 meniscus and articular cartilage and 43 genes were differentially expressed between E16 meniscus and cruciate ligaments. Included in our data set were extracellular matrix proteins, transcription factors, and growth factors, including TGF‐β modulators (Lox, Dpt) and IGF‐1 pathway members (Igf‐1, Igfbp2, Igfbp3, Igfbp5). Ingenuity Pathway Analysis revealed that IGF‐1 signaling was enriched in the meniscus compared to the other joint structures, while qPCR showed that Igf‐1, Igfbp2, and Igfbp3 expression declined with age. We also found that several meniscus‐enriched genes were expressed either in the inner or outer meniscus, establishing that regionalization of the meniscus occurs early in development.

Specification of osteoblast cell fate by canonical Wnt signaling requires Bmp2

Enhanced BMP or canonical Wnt (cWnt) signaling are therapeutic strategies employed to enhance bone formation and fracture repair, but the mechanisms each pathway utilizes to specify cell fate of bone-forming osteoblasts remain poorly understood. Among all BMPs expressed in bone, we find that singular deficiency of Bmp2 blocks the ability of cWnt signaling to specify osteoblasts from limb bud or bone marrow progenitors. When exposed to cWnts, Bmp2-deficient cells fail to progress through the Runx2/Osx1 checkpoint and thus do not upregulate multiple genes controlling mineral metabolism in osteoblasts. Cells lacking Bmp2 after induction of Osx1 differentiate normally in response to cWnts, suggesting that pre-Osx1+ osteoprogenitors are an essential source and a target of BMP2. Our analysis furthermore reveals Grainyhead-like 3 (Grhl3) as a transcription factor in the osteoblast gene regulatory network induced during bone development and bone repair, which acts upstream of Osx1 in a BMP2-dependent manner. The Runx2/Osx1 transition therefore receives crucial regulatory inputs from BMP2 that are not compensated for by cWnt signaling, and this is mediated at least in part by induction and activation of Grhl3. Summary: Osteoblast specification requires regulatory inputs from BMP2 at the Runx2/Osx1 transition by a mechanism that is not compensated for by canonical Wnt signaling and involves Grhl3.