scholarly journals GNAS Heterozygous Inactivation Differentially Affects Osteoclast-Specific Calcitonin Receptor Bioactivity in a Mouse Model of Albright Hereditary Osteodystrophy Based Upon Parental Inheritance

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A687-A688
Author(s):  
Patrick McMullan ◽  
Peter Maye ◽  
Qingfen Yang ◽  
David Rowe ◽  
Emily L Germain-Lee
Keyword(s):  
Mouse Model ◽  
Mrna Expression ◽  
Fluorescent Microscopy ◽  
Receptor Activity ◽  
Pcr Analysis ◽  
Mineral Density ◽  
Actin Ring ◽  
Albright Hereditary Osteodystrophy ◽  
Serum Pth ◽  
Parental Inheritance

Abstract Albright hereditary osteodystrophy (AHO) is caused by the heterozygous inactivation of GNAS, encoding the α-stimulatory subunit (Gαs) of G protein-coupled receptors. Skeletal manifestations of AHO include adult short stature, brachydactyly and subcutaneous ossifications. AHO patients with maternally-derived GNAS mutations develop pseudohypoparathyroidism type 1A (PHP1A) and are obese with resistance to hormones requiring Gαs (eg., PTH, TSH and GHRH) due to tissue-specific GNAS imprinting. Paternally-derived GNAS mutations cause pseudopseudohypoparathyroidism (PPHP) in which patients have AHO skeletal features but do not develop severe obesity or hormonal resistance. Mouse models have shown loss of Gα s signaling in osteoblasts or osteoclasts leads to osteopenia, and suggest AHO patients would display a reduced bone mineral density (BMD). Interestingly, PHP1A patients have been shown to have normal to increased BMD without any correlation to body mass index or serum PTH measurement. Based on the differences observed clinically and hormonally between PHP1A and PPHP, we hypothesize that there may also be distinctions in overall bone remodeling between these two disorders due to GNAS imprinting. This study addressed whether the heterozygous inactivation of Gnas differentially affects Gα s-receptor bioactivity within osteoclasts (OCs) based upon parental inheritance. Bone Marrow Macrophages (BMMs) were harvested from our laboratory’s AHO mouse model with either maternally-inherited (Gnas+/-m) mutations correlating to PHP1A or paternally-inherited (Gnas+/-p) mutations correlating to PPHP. BMMs were exposed to 10-7M salmon calcitonin (sCT), 10-5M forskolin or PBS for 6 hrs. OC receptor activity was measured by fluorescent microscopy to visualize actin ring morphology and RT-PCR analysis of Gα s-PKA signaling transcripts Crem and Ramp3. Forskolin treatment displayed no significant variations in OC ring morphology or Crem and Ramp3 mRNA expression between Gnas+/-m, Gnas+/-p and WT cultures. Both WT and Gnas+/-p OCs displayed appropriate responses to sCT, as indicated by a significant disruption in actin ring morphology and increased Crem and Ramp3 mRNA expression when compared to vehicle-treated controls. SCT-treated Gnas+/-m OCs, however, displayed only mild disruptions in actin ring morphology, and we observed significant reductions in Ramp3 expression compared to WT as well as reductions in Crem compared to WT and Gnas+/-p. These data suggest evidence of partial calcitonin resistance within Gnas+/-m OCs due to impaired Gα s- signaling. These data correlate with previous clinical observations of calcitonin resistance in PHP1A patients. Because these findings were observed only within Gnas+/-m cultures, future work is warranted to determine whether this impaired receptor activity may be attributed to partial Gnas imprinting within OCs or the myeloid lineage.

scholarly journals Parental origin of Gsα inactivation differentially affects bone remodeling in a mouse model of Albright hereditary osteodystrophy

2021 ◽  
Author(s):  
Patrick McMullan ◽  
Qingfen Yang ◽  
Peter Maye ◽  
David Rowe ◽  
Emily Germain-Lee
Keyword(s):  
Bone Resorption ◽  
Mouse Model ◽  
Bone Remodeling ◽  
Parental Origin ◽  
Mineral Density ◽  
Bone Parameters ◽  
Albright Hereditary Osteodystrophy ◽  
Osteoblast Activity ◽  
Hormonal Resistance ◽  
Serum Pth

Albright hereditary osteodystrophy (AHO) is caused by heterozygous inactivation of GNAS, a complex locus that encodes the alpha-stimulatory subunit of GPCRs (Gsα) in addition to NESP55 and XLαs due to alternative first exons. AHO skeletal manifestations include brachydactyly, brachymetacarpia, compromised adult stature, and subcutaneous ossifications. AHO patients with maternally-inherited GNAS mutations develop pseudohypoparathyroidism type 1A (PHP1A) with resistance to multiple hormones that mediate their actions through GPCRs requiring Gsα (eg., PTH, TSH, GHRH, calcitonin) and severe obesity. Paternally-inherited GNAS mutations cause pseudopseudohypoparathyroidism (PPHP), in which patients have AHO skeletal features but do not develop hormonal resistance or marked obesity. These differences between PHP1A and PPHP are caused by tissue-specific reduction of paternal Gsα expression. Previous reports in mice have shown loss of Gsα causes osteopenia due to impaired osteoblast number and function and suggest AHO patients could display evidence of reduced bone mineral density (BMD). However, we previously demonstrated PHP1A patients display normal-increased BMD measurements without any correlation to body mass index or serum PTH. Due to these observed differences between PHP1A and PPHP, we utilized an AHO mouse model generated in our laboratory to address whether Gsα heterozygous inactivation by the targeted disruption of exon 1 of Gnas differentially affects bone remodeling based on the parental inheritance of the mutation. Mice with paternally-inherited (Gnas E1+/-p) and maternally-inherited (Gnas E1+/-m) mutations displayed reductions in osteoblasts along the bone surface compared to wildtype. Gnas E1+/-p mice displayed reduced cortical and trabecular bone parameters due to impaired bone formation and excessive bone resorption. Gnas E1+/-m mice however displayed enhanced bone parameters due to increased osteoblast activity and normal bone resorption. These distinctions in bone remodeling between Gnas E1+/-p and Gnas E1+/-m mice appear to be secondary to changes in the bone microenvironment driven by calcitonin-resistance within Gnas E1+/-m osteoclasts and therefore warrant further studies into understanding how Gsα influences osteoblast-osteoclast coupling interactions.

scholarly journals Regulation of bone phosphorus retention and bone development possibly by related hormones and local bone-derived regulators in broiler chicks

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sumei Cao ◽  
Tingting Li ◽  
Yuxin Shao ◽  
Liyang Zhang ◽  
Lin Lu ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Bone Mineral ◽  
Matrix Protein ◽  
Bone Development ◽  
Fibroblast Growth Factor 23 ◽  
Phosphorus Retention ◽  
Mineral Density ◽  
Local Bone ◽  
Serum Pth ◽  
Tibia Ash

Abstract Background Phosphorus is essential for bone mineralization in broilers, however, the underlying mechanisms remain unclear. We aimed to investigate whether bone phosphorus retention and bone development might be regulated by related hormones and local bone-derived regulators in broilers. Methods Broilers were fed diets containing different levels of non-phytate phosphorus (NPP) 0.15%, 0.25%, 0.35%, 0.45% and 0.55% or 0.15%, 0.22%, 0.29%, 0.36% and 0.43% from 1 to 21 or 22 to 42 days of age. Serum and tibia samples were collected for determinations of bone phosphorus retention and bone development parameters, related hormones and local bone-derived regulators of broiler chickens on d 14, 28 and 42, respectively. Results Tibia ash phosphorus, total phosphorus accumulation in tibia ash (TPTA), bone mineral concentration (BMC), bone mineral density (BMD), bone breaking strength (BBS), and ash on d 14, 28 or 42, serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on d 28 and 42, mRNA expressions of tibia fibroblast growth factor 23 (FGF23) and dentin matrix protein 1 (DMP1) on d 14 and 28 increased linearly or quadratically (P < 0.05), while serum parathyroid hormone (PTH) on d 28, tibia alkaline phosphatase (ALP) on d 14, 28 and 42, bone gal protein (BGP) on d 14, and mRNA expression of tibia phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) on d 14 and 28 decreased linearly or quadratically (P < 0.04) as dietary NPP level increased. TPTA, BMC, BMD, and ash on d 28 and 42, BBS on d 28, and ash phosphorus on d 42 were positively correlated (r = 0.389 to 0.486, P < 0.03) with serum 1,25(OH)2D3. All of the above parameters were positively correlated (r = 0.380 to 0.689, P < 0.05) with tibia DMP1 mRNA expression on d 14, 28 and 42, but negatively correlated (r = − 0.609 to − 0.538, P < 0.02) with serum PTH on d 28, tibia ALP on d 14, 28 and 42, and BGP on d 14. TPTA, BMC and ash on d 14 and BMD on d 28 were negatively correlated (r = − 0.397 to − 0.362, P < 0.03) with tibia PHEX mRNA expression, and BMD on d 28 was positively correlated (r = 0.384, P = 0.04) with tibia FGF23 mRNA expression. Conclusions These results suggested that bone phosphorus retention and bone development parameters had moderate to strong correlations with serum PTH and 1,25(OH)2D3 and tibia DMP1, PHEX, FGF23, ALP and BGP in broilers during the whole growth period, and thus they might be partly regulated by these related hormones and local bone-derived regulators.

Healing effects of curcumin nanoparticles in deep tissue injury mouse model.

2020 ◽  
Vol 18 ◽  
Author(s):  
Zirui Zhang ◽  
Shangcong Han ◽  
Panpan Liu ◽  
Xu Yang ◽  
Jing Han ◽  
...  
Keyword(s):  
Wound Healing ◽  
Mrna Expression ◽  
Tissue Injury ◽  
Inflammatory Cells ◽  
Pcr Analysis ◽  
Protective Effects ◽  
Deep Tissue ◽  
Deep Tissue Injury

Background: Chronic inflammation and lack of angiogenesis are the important pathological mechanisms in deep tissue injury (DTI). Curcumin is a well-known anti-inflammatory and antioxidant agent. However, curcumin is unstable under acidic and alkaline conditions, and can be rapidly metabolized and excreted in the bile, which shortens its bioactivity and efficacy. Objective: This study aimed to prepare curcumin-loaded poly (lactic-co-glycolic acid) nanoparticles (CPNPs) and to elucidate the protective effects and underlying mechanisms of wound healing in DTI models. Methods: CPNPs were evaluated for particle size, biocompatibility, in vitro drug release and their effect on in vivo wound healing. Results : The results of in vivo wound closure analysis revealed that CPNP treatments significantly improved wound contraction rates (p<0.01) at a faster rate than other three treatment groups. H&E staining revealed that CPNP treatments resulted in complete epithelialization and thick granulation tissue formation, whereas control groups resulted in a lack of compact epithelialization and persistence of inflammatory cells within the wound sites. Quantitative real-time PCR analysis showed that treatment with CPNPs suppressed IL-6 and TNF-α mRNA expression, and up-regulated TGF-β, VEGF-A and IL-10 mRNA expression. Western blot analysis showed up-regulated protein expression of TGF-β, VEGF-A and phosphorylatedSTAT3. Conclusion: Our results showed that CPNPs enhanced wound healing in DTI models, through modulation of the JAK2/STAT3 signalling pathway and subsequent upregulation of pro-healing factors.

Quantitative RT-PCR analysis of MMP and TIMP MRNA expression in correlation to liver histology in patients with chronic hepatitis C

2000 ◽  
Vol 118 (4) ◽  
pp. A1469
Author(s):  
Dirk Michels ◽  
Christian I. Haberkorn ◽  
Burkhard Arndt ◽  
Michael P. Manns
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C ◽  
Mrna Expression ◽  
Chronic Hepatitis C ◽  
Liver Histology ◽  
Pcr Analysis ◽  
Rt Pcr

scholarly journals Hepatic Glucocorticoid Receptor Plays a Greater Role Than Adipose GR in Metabolic Syndrome Despite Renal Compensation

Endocrinology ◽  
2016 ◽  
Vol 157 (12) ◽  
pp. 4943-4960 ◽  
Author(s):  
Sandip K. Bose ◽  
Irina Hutson ◽  
Charles A. Harris
Keyword(s):  
Metabolic Syndrome ◽  
Glucocorticoid Receptor ◽  
Mrna Expression ◽  
Kidney Tissue ◽  
Glycogen Metabolism ◽  
Whole Body ◽  
Pcr Analysis ◽  
Compensatory Mechanism ◽  
Key Genes ◽  
Specific Deletion

Exogenous glucocorticoid administration results in hyperglycemia, insulin resistance, hepatic dyslipidemia, and hypertension, a constellation of findings known as Cushing’s syndrome. These effects are mediated by the glucocorticoid receptor (GR). Because GR activation in liver and adipose has been implicated in metabolic syndrome (MS), we wanted to determine the role of GR in these tissues in the development of MS. Because GR knockout (KO) mice (whole-body KO) exhibit perinatal lethality due to respiratory failure, we generated tissue-specific (liver or adipose) GRKO mice using cre-lox technology. Real-time PCR analysis of liver mRNA from dexamethasone-treated wildtype (WT) and liver GRKO mice indicated that hepatic GR regulates the expression of key genes involved in gluconeogenesis and glycogen metabolism. Interestingly, we have observed that liver-specific deletion of GR resulted in a significant increase in mRNA expression of key genes involved in gluconeogenesis and glycogen metabolism in kidney tissue, indicating a compensatory mechanism to maintain glucose homeostasis. We have also observed that GR plays an important role in regulating the mRNA expression of key genes involved in lipid metabolism. Liver GRKO mice demonstrated decreased fat mass and liver glycogen content compared with WT mice administered dexamethasone for 2 weeks. Adipose-specific deletion of GR did not alter glucose tolerance or insulin sensitivity of adipose GRKO mice compared with WT mice administrated dexamethasone. This indicates that liver GR might be more important in development of MS in dexamethasone-treated mice, whereas adipose GR plays a little role in these paradigms.

Free fall landing and interval running have different effects on trabecular bone mass and microarchitecture, serum osteocalcin, biomechanical properties, SOST expression and on osteocyte related characteristics

2021 ◽  
Author(s):  
Arnaud Boudenot ◽  
stephane PALLU ◽  
Rustem UZBEKOV ◽  
Eric DOLLEANS ◽  
Hechmi Toumi ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Mrna Expression ◽  
Interval Training ◽  
Free Fall ◽  
Biomechanical Properties ◽  
Bending Test ◽  
Blue Staining ◽  
Control Group ◽  
Mineral Density ◽  
Osteocyte Lacunae

The effects of treadmill interval training (IT) and free fall exercise were evaluated on bone parameters including osteocyte related characteristics. Thirty-eight 4-month-old male Wistar rats were randomly divided into a control group (C) and exercise groups: IT, 10 free fall impacts/day with a 10s (FF10) or 20s interval between drops (FF20), 5 days/week, for 9 weeks. We assessed: BMD, microarchitecture by µCT, mechanical strength by a three-point bending test, density and occupancy of the osteocyte lacunae by toluidine blue staining, osteocalcin and NTx systemic levels by ELISA, and bone tissue Sost mRNA expression by RT-PCR. NTx levels were significantly lower in exercise groups as compared to C. In exercise groups Sost mRNA expression was significantly lower than in C. Tb.N was significantly higher for IT and FF20 compared to C; Tb.Sp was significantly lower in FF10 compared to C. Both IT and FF20 were associated with higher tibial lacunar density as compared to FF10. Compared to FF10, IT fat mass was lower, while tibial osteocyte lacunae occupancy and systemic osteocalcin level were higher. All exercise modes were efficient in reducing bone resorption. Both IT and FF impact with appropriate recovery periods might be beneficial for bone health and osteocyte related characteristics. Novelty bullets: • Interval training is beneficial for bone mineral density • Exercises decreased both bone resorption and inhibition of bone formation (sost mRNA) • Longer interval recovery time favors osteocyte lacunae density

scholarly journals Mouse Model of Loeys–Dietz Syndrome Shows Elevated Susceptibility to Periodontitis via Alterations in Transforming Growth Factor-Beta Signaling

2021 ◽  
Vol 12 ◽  
Author(s):  
Satoru Yamada ◽  
Kenichiro Tsushima ◽  
Masaki Kinoshita ◽  
Hiromi Sakashita ◽  
Tetsuhiro Kajikawa ◽  
...  
Keyword(s):  
Growth Factor ◽  
Bone Loss ◽  
Mouse Model ◽  
Mrna Expression ◽  
Missense Mutation ◽  
Transforming Growth Factor ◽  
Alveolar Bone ◽  
Elastic Fibers ◽  
Periodontal Tissues ◽  
Significant Difference

Loeys–Dietz syndrome (LDS) is a syndromic connective tissue disorder caused by a heterozygous missense mutation in genes that encode transforming growth factor (TGF)-β receptor (TGFBR) 1 and 2. We encountered a patient with LDS, who had severe periodontal tissue destruction indicative of aggressive periodontitis. The patient had a missense mutation in the glycine and serine-rich domain of TGFBR1 exon 3. This G-to-T mutation at base 563 converted glycine to valine. We established an LDS model knock-in mouse that recapitulated the LDS phenotype. Homozygosity of the mutation caused embryonic lethality and heterozygous knock-in mice showed distorted and ruptured elastic fibers in the aorta at 24 weeks of age and died earlier than wildtype (WT) mice. We stimulated mouse embryonic fibroblasts (MEFs) from the knock-in mouse with TGF-β and examined their responses. The knock-in MEFs showed downregulated Serpine 1 mRNA expression and phosphorylation of Smad2 to TGF-β compared with WT MEFs. To clarify the influence of TGF-β signaling abnormalities on the pathogenesis or progression of periodontitis, we performed pathomolecular analysis of the knock-in mouse. There were no structural differences in periodontal tissues between WT and LDS model mice at 6 or 24 weeks of age. Micro-computed tomography revealed no significant difference in alveolar bone resorption between WT and knock-in mice at 6 or 24 weeks of age. However, TGF-β-related gene expression was increased significantly in periodontal tissues of the knock-in mouse compared with WT mice. Next, we assessed a mouse periodontitis model in which periodontal bone loss was induced by oral inoculation with the bacterial strain Porphyromonas gingivalis W83. After inoculation, we collected alveolar bone and carried out morphometric analysis. P. gingivalis-induced alveolar bone loss was significantly greater in LDS model mice than in WT mice. Peritoneal macrophages isolated from Tgfbr1G188V/+ mice showed upregulation of inflammatory cytokine mRNA expression induced by P. gingivalis lipopolysaccharide compared with WT macrophages. In this study, we established an LDS mouse model and demonstrated that LDS model mice had elevated susceptibility to P. gingivalis-induced periodontitis, probably through TGF-β signal dysfunction. This suggests that TGF-β signaling abnormalities accelerate the pathogenesis or progression of periodontitis.

scholarly journals Anabolic Actions of Parathyroid Hormone during Bone Growth Are Dependent on c-fos

Endocrinology ◽  
2002 ◽  
Vol 143 (10) ◽  
pp. 4038-4047 ◽  
Author(s):  
Burak Demiralp ◽  
Hen-Li Chen ◽  
Amy J. Koh ◽  
Evan T. Keller ◽  
Laurie K. McCauley
Keyword(s):  
Trabecular Bone ◽  
Mrna Expression ◽  
Bone Growth ◽  
Critical Role ◽  
Calcium Content ◽  
Wild Type ◽  
Mineral Density ◽  
Femoral Bone Mineral Density ◽  
Endochondral Bone ◽  
Ash Weight

Abstract PTH has anabolic and catabolic actions in bone that are not clearly understood. The protooncogene c-fos and other activating protein 1 family members are critical transcriptional mediators in bone, and c-fos is up-regulated by PTH. The purpose of this study was to examine the mechanisms of PTH and the role of c-fos in PTH-mediated anabolic actions in bone. Mice with ablation of c-fos (−/−) and their wild-type (+/+) and heterozygous (+/−) littermates were administered PTH for 17 d. The +/+ mice had increased femoral bone mineral density (BMD), whereas −/− mice had reduced BMD after PTH treatment. PTH increased the ash weight of +/+ and +/−, but not −/−, femurs and decreased the calcium content of −/−, but not +/+ or +/−, femurs. Histomorphometric analysis showed that PTH increased trabecular bone volume in c-fos +/+, +/− vertebrae, but, in contrast, decreased trabecular bone in −/− vertebrae. Serum calcium levels in +/+ mice were greater than those in −/− mice, and PTH increased calcium in −/− mice. Histologically, PTH resulted in an exacerbation of the already widened growth plate and zone of hypertrophic chondrocytes but not the proliferating zone in −/− mice. PTH also increased calvarial thickness in +/+ mice, but not −/− mice. The c-fos −/− mice had lower bone sialoprotein and osteocalcin (OCN), but unaltered PTH-1 receptor mRNA expression in calvaria, suggesting an alteration in extracellular matrix. Acute PTH injection (8 h) resulted in a decrease in osteocalcin mRNA expression in wild-type, but unaltered expression in −/−, calvaria. These data indicate that c-fos plays a critical role in the anabolic actions of PTH during endochondral bone growth.

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