Girk3 deletion increases osteoblast maturation and bone mass accrual in adult male mice

AUTHORS

Samantha R Weaver, Haydee M Torres, Katherine M Arnold, Elizabeth L Zars, Eduardo Peralta-Herrera, Earnest L Taylor, Kanglun Yu, Ezequiel Marron Fernandez de Velasco, Kevin Wickman, Meghan E McGee-Lawrence, Elizabeth W Bradley, Jennifer J Westendorf

ABSTRACT

Osteoporosis and other metabolic bone diseases are prevalent in the aging population. While bone has the capacity to regenerate throughout life, bone formation rates decline with age and contribute to reduced bone density and strength. Identifying mechanisms and pathways that increase bone accrual in adults could prevent fractures and accelerate healing. G protein-gated inwardly rectifying K+ (GIRK) channels are key effectors of G protein-coupled receptor signaling. Girk3 was recently shown to regulate endochondral ossification. Here, we demonstrate that deletion of Girk3 increases bone mass after 18 weeks of age. Male 24-week-old Girk3-/- mice have greater trabecular bone mineral density and bone volume fraction than wildtype (WT) mice. Osteoblast activity is moderately increased in 24-week-old Girk3-/- mice compared to WT mice. In vitro, Girk3-/- bone marrow stromal cells (BMSCs) are more proliferative than WT BMSCs. Calvarial osteoblasts and BMSCs from Girk3-/- mice are also more osteogenic than WT cells, with altered expression of genes that regulate the wingless-related integration site (Wnt) family. Wnt inhibition via Dickkopf-1 (Dkk1) or β-catenin inhibition via XAV939 prevents enhanced mineralization, but not proliferation, in Girk3-/- BMSCs and slows these processes in WT cells. Finally, selective ablation of Girk3 from cells expressing Cre recombinase from the 2.3 kb-Col1a1 promoter, including osteoblasts and osteocytes, is sufficient to increase bone mass and bone strength in male mice at 24 weeks of age. Taken together, these data demonstrate that Girk3 regulates progenitor cell proliferation, osteoblast differentiation, and bone mass accrual in adult male mice.

Prenatal protein malnutrition decreases neuron numbers in the parahippocampal region but not prefrontal cortex in adult rats

AUTHORS

A. C. Amaral, J. P. Lister, J. W. Rueckemann, M. W. Wojnarowicz, J. A. McGaughy, D. J. Mokler, J. R. Galler, D. L. Rosene, R. J. Rushmore

ABSTRACT

Objective

Prenatal protein malnutrition produces anatomical and functional changes in the developing brain that persist despite immediate postnatal nutritional rehabilitation. Brain networks of prenatally malnourished animals show diminished activation of prefrontal areas and an increased activation of hippocampal regions during an attentional task [1]. While a reduction in cell number has been documented in hippocampal subfield CA1, nothing is known about changes in neuron numbers in the prefrontal or parahippocampal cortices.

Methods

In the present study, we used unbiased stereology to investigate the effect of prenatal protein malnutrition on the neuron numbers in the medial prefrontal cortex and the cortices of the parahippocampal region that comprise the larger functional network.

Results

Results show that prenatal protein malnutrition does not cause changes in the neuronal population in the medial prefrontal cortex of adult rats, indicating that the decrease in functional activation during attentional tasks is not due to a reduction in the number of neurons. Results also show that prenatal protein malnutrition is associated with a reduction in neuron numbers in specific parahippocampal subregions: the medial entorhinal cortex and presubiculum.

Discussion

The affected regions along with CA1 comprise a tightly interconnected circuit, suggesting that prenatal malnutrition confers a vulnerability to specific hippocampal circuits. These findings are consistent with the idea that prenatal protein malnutrition produces a reorganization of structural and functional networks, which may underlie observed alterations in attentional processes and capabilities.

Multiscale effects of the calcimimetic drug, etelcalcetide on bone health of rats with secondary hyperparathyroidism induced by chronic kidney disease

AUTHORS

Shivani Sharma, Saroj Kumar, Manendra Singh Tomar, Divya Chauhan, Chirag Kulkarni, Swati Rajput, Sreyanko Sadhukhan, Konica Porwal, Rajdeep Guha, Ashutosh Shrivastava, Jiaur R. Gayen, Navin Kumar, Naibedya Chattopadhyay

ABSTRACT

Chronic kidney disease-induced secondary hyperparathyroidism (CKD-SHPT) heightens fracture risk through impaired mineral homeostasis and elevated levels of uremic toxins (UTs), which in turn enhance bone remodeling. Etelcalcetide (Etel), a calcium-sensing receptor (CaSR) agonist, suppresses parathyroid hormone (PTH) in hyperparathyroidism to reduce excessive bone resorption, leading to increased bone mass. However, Etel's effect on bone quality, chemical composition, and strength is not well understood. To address these gaps, we established a CKD-SHPT rat model and administered Etel at a human-equivalent dose concurrently with disease induction. The effects on bone and mineral homeostasis were compared with a CKD-SHPT (vehicle-treated group) and a control group (rats without SHPT). Compared with vehicle-treated CKD-SHPT rats, Etel treatment improved renal function, reduced circulating UT levels, improved mineral homeostasis parameters, decreased PTH levels, and prevented mineralization defects. The upregulation of mineralization-promoting genes by Etel in CKD-SHPT rats might explain its ability to prevent mineralization defects. Etel preserved both trabecular and cortical bones with attendant suppression of osteoclast function, besides increasing mineralization. Etel maintained the number of viable osteocytes to the control level, which could also contribute to its beneficial effects on bone. CKD-SHPT rats displayed increased carbonate substitution of matrix and mineral, decreased crystallinity, mineral-to-matrix ratio, and collagen maturity, and these changes were mitigated by Etel. Further, Etel treatment prevented CKD-SHPT-induced deterioration in bone strength and mechanical behavior. Based on these findings, we conclude that in CKD-SHPT rats, Etel has multiscale beneficial effects on bone that involve remodeling suppression, mineralization gene upregulation, and preservation of osteocytes.

Loss of the systemic vitamin A transporter RBPR2 affects the quantitative balance between chromophore and opsins in visual pigment synthesis

AUTHORS

Rakesh Radhakrishnan, Matthias Leung, Anjelynt Lor, Swati More, Glenn P Lobo

ABSTRACT

The distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and for generating visual pigments for photoreceptor cell function. ROL circulates in the blood bound to the retinol binding protein 4 (RBP4) as RBP4-ROL. Two membrane receptors, RBPR2 in the liver and STRA6 in the eye are proposed to bind circulatory RBP4 and this mechanism is critical for internalizing ROL into cells. Here, we present a longitudinal investigation towards the importance of RBPR2 and influence of the diet on systemic retinoid homeostasis for visual function. Age matched Rbpr2-KO (Rbpr2−/−) and wild-type (WT) mice were fed either a vitamin A sufficient (VAS) or a vitamin A deficient (VAD) diet. At 3- and 6-months, we performed retinoid quantification of ocular and non-ocular tissues using HPLC analysis and complemented the data with visual physiology, rhodopsin quantification by spectrophotometry, and biochemical analysis. At 3-months and compared to WT mice, Rbpr2−/− mice fed either vitamin A diets displayed lower scotopic and photopic electroretinogram (ERG) responses, which correlated with HPLC analysis that revealed Rbpr2−/− mice had significantly lower hepatic and ocular retinoid content. Interestingly, with the exception of the liver, long-term feeding of Rbpr2−/− mice with a VAS diet promoted all-trans retinol accumulation in most peripheral tissues. However, even under VAS dietary conditions significant amounts of unliganded opsins in rods, together with decreased visual responses were evident in aged mice lacking RBPR2, when compared to WT mice. Together, our analyses characterize the molecular events underlying nutritional blindness in a novel mouse model and indicate that loss of the liver specific RBP4-ROL receptor, RBPR2, influences systemic retinoid homeostasis and rhodopsin synthesis, which causes profound visual function defects under severe vitamin A deficiency conditions.

Osteoblast-specific down-regulation of NLRP3 inflammasome by aptamer-functionalized liposome nanoparticles improves bone quality in postmenopausal osteoporosis rats

AUTHORS

Lijun Xu, Jie Zhu, Lingjun Rong, Huinan Yang, Bin Wang, Shuai Lu, Lingxiao Zhang, Fuyi Li, Shihua Yang, Zhifang Wang, Chong Li, Xiao Hu, Ruoyun Liu, Lili Zheng, Hongjian Liu, Haohao Zhang, Yanling Liu, Di Zhao, Shuiying Zhao, Lun Zhang, Yingbo Jia, Shiyu Liang, Zhikang Guo, Xixiu Xi, Ruitian Liu, Lixia Zhang

ABSTRACT

Rationale: NLRP3 inflammasome is critical in the development and progression of many metabolic diseases driven by chronic inflammation, but its effect on the pathology of postmenopausal osteoporosis (PMOP) remains poorly understood.

Methods: We here firstly examined the levels of NLRP3 inflammasome in PMOP patients by ELISA. Then we investigated the possible mechanisms underlying the effect of NLRP3 inflammasome on PMOP by RNA sequencing of osteoblasts treated with NLRP3 siRNA and qPCR. Lastly, we accessed the effect of decreased NLRP3 levels on ovariectomized (OVX) rats. To specifically deliver NLRP3 siRNA to osteoblasts, we constructed NLRP3 siRNA wrapping osteoblast-specific aptamer (CH6)-functionalized lipid nanoparticles (termed as CH6-LNPs-siNLRP3).

Results: We found that the levels of NLRP3 inflammasome were significantly increased in patients with PMOP, and were negatively correlated with estradiol levels. NLRP3 knock-down influenced signal pathways including immune system process, interferon signal pathway. Notably, of the top ten up-regulated genes in NLRP3-reduced osteoblasts, nine genes (except Mx2) were enriched in immune system process, and five genes were related to interferon signal pathway. The in vitro results showed that CH6-LNPs-siNLRP3 was relatively uniform with a dimeter of 96.64 ± 16.83 nm and zeta potential of 38.37 ± 1.86 mV. CH6-LNPs-siNLRP3 did not show obvious cytotoxicity and selectively delivered siRNA to bone tissue. Moreover, CH6-LNPs-siNLRP3 stimulated osteoblast differentiation by activating ALP and enhancing osteoblast matrix mineralization. When administrated to OVX rats, CH6-LNPs-siNLRP3 promoted bone formation and bone mass, improved bone microarchitecture and mechanical properties by decreasing the levels of NLRP3, IL-1β and IL-18 and increasing the levels of OCN and Runx2.

Conclusion: NLRP3 inflammasome may be a new biomarker for PMOP diagnosis and plays a key role in the pathology of PMOP. CH6-LNPs-siNLRP3 has potential application for the treatment of PMOP.

Two Weeks of Continuous Opioid Treatment in an Adenine-Induced Mouse Model of Chronic Kidney Disease Exacerbates the Bone Inflammatory State and Increases Osteoclasts

AUTHORS

Corinne E. Metzger, Gregory G. Grecco, Landon Y. Tak, Brady K. Atwood & Matthew R. Allen

ABSTRACT

Patients with chronic kidney disease (CKD) report high pain levels, but reduced renal clearance eliminates many analgesic options; therefore, 30–50% of CKD patients have chronic opioid prescriptions. Opioid use in CKD is associated with higher fracture rates. Opioids may directly alter bone turnover directly through effects on bone cells and indirectly via increasing inflammation. We hypothesized that continuous opioid exposure would exacerbate the high bone turnover state of CKD and be associated with elevated measures of inflammation. Male C57Bl/6J mice after 8 weeks of adenine-induced CKD (AD) and non-AD controls (CON) had 14-day osmotic pumps (0.25-µL/hr release) containing either saline or 50-mg/mL oxycodone (OXY) surgically implanted in the subscapular region. After 2 weeks, all AD mice had elevated blood urea nitrogen, parathyroid hormone, and serum markers of bone turnover compared to controls with no effect of OXY. Immunohistochemical staining of the distal femur showed increased numbers of osteocytes positive for the mu opioid and for toll-like receptor 4 (TLR4) due to OXY. Osteocyte protein expression of tumor necrosis factor-α (TNF-α) and RANKL were higher due to both AD and OXY so that AD + OXY mice had the highest values. Trabecular osteoclast-covered surfaces were also significantly higher due to both AD and OXY, resulting in AD + OXY mice having 4.5-fold higher osteoclast-covered surfaces than untreated CON. These data demonstrate that opioids are associated with a pro-inflammatory state in osteocytes which increases the pro-resorptive state of CKD.