Prkd1 regulates the formation and repair of plasma membrane disruptions (PMD) in osteocytes

AUTHORS

Anik Tuladhar, Joseph C. Shaver, Wesley A. McGee, Kanglun Yu, Jennifer Dorn, J. Luke Horne, Dima W. Alhamad, Mackenzie L. Hagan, Marion A. Cooley, Roger Zhong, Wendy Bollag, Maribeth Johnson, Mark W. Hamrick, Meghan E. McGee-Lawrence

ABSTRACT

We and others have seen that osteocytes sense high-impact osteogenic mechanical loading via transient plasma membrane disruptions (PMDs) which initiate downstream mechanotransduction. However, a PMD must be repaired for the cell to survive this wounding event. Previous work suggested that the protein Prkd1 (also known as PKCμ) may be a critical component of this PMD repair process, but the specific role of Prkd1 in osteocyte mechanobiology had not yet been tested. We treated MLO-Y4 osteocytes with Prkd1 inhibitors (Go6976, kbNB 142-70, staurosporine) and generated an osteocyte-targeted (Dmp1-Cre) Prkd1 conditional knockout (CKO) mouse. PMD repair rate was measured via laser wounding and FM1-43 dye uptake, PMD formation and post-wounding survival were assessed via fluid flow shear stress (50 dyn/cm2), and in vitro osteocyte mechanotransduction was assessed via measurement of calcium signaling. To test the role of osteocyte Prkd1 in vivo, Prkd1 CKO and their wildtype (WT) littermates were subjected to 2 weeks of unilateral axial tibial loading and loading-induced changes in cortical bone mineral density, geometry, and formation were measured.

Prkd1 inhibition or genetic deletion slowed osteocyte PMD repair rate and impaired post-wounding cell survival. These effects could largely be rescued by treating osteocytes with the FDA-approved synthetic copolymer Poloxamer 188 (P188), which was previously shown to facilitate membrane resealing and improve efficiency in the repair rate of PMD in skeletal muscle myocytes. In vivo, while both WT and Prkd1 CKO mice demonstrated anabolic responses to tibial loading, the magnitude of loading-induced increases in tibial BMD, cortical thickness, and periosteal mineralizing surface were blunted in Prkd1 CKO as compared to WT mice. Prkd1 CKO mice also tended to show a smaller relative difference in the number of osteocyte PMD in loaded limbs and showed greater lacunar vacancy, suggestive of impaired post-wounding osteocyte survival. While P188 treatment rescued loading-induced increases in BMD in the Prkd1 CKO mice, it surprisingly further suppressed loading-induced increases in cortical bone thickness and cortical bone formation. Taken together, these data suggest that Prkd1 may play a pivotal role in the regulation and repair of the PMD response in osteocytes and support the idea that PMD repair processes can be pharmacologically targeted to modulate downstream responses, but suggest limited utility of PMD repair-promoting P188 in improving bone anabolic responses to loading.

Effects of Short-Term Treatment of Rabbit Extraocular Muscle With Ciliary Neurotrophic Factor

AUTHORS

Jolene C. Rudell; Linda K. McLoon

ABSTRACT

Purpose: Little is known about the effect of ciliary neurotrophic factor (CNTF) on extraocular muscles, but microarray studies suggested CNTF might play a role in the development and/or maintenance of strabismus. The effect of short-term treatment of adult rabbit extraocular muscle with injected CNTF was examined for its ability to alter muscle characteristics.

Methods: Eight adult New Zealand white rabbits received an injection into one superior rectus muscle of 2 µg/100 µL CNTF on 3 consecutive days. One week after the first injection, the rabbits were euthanized, and the treated and contralateral superior rectus muscles were assessed for force generation capacity and contraction characteristics using an in vitro stimulation protocol and compared to naïve control superior rectus muscles. All muscles were analyzed to determine mean cross-sectional areas and expression of slow twitch myosin heavy chain isoform.

Results: Short-term treatment of rabbit superior rectus muscles with CNTF resulted in a significant decrease in muscle force generation, but only at the higher stimulation frequencies. Significantly decreased myofiber cross-sectional areas of the treated muscles correlated with the decreased generated force. In addition, there were significant changes to contractile properties of the treated muscles, as well as a decrease in the number of myofibers expressing slow twitch myosin heavy chain.

Conclusions: We show that short-term treatment of a single rabbit superior rectus muscle results in decreased myofiber size, decreased force, and altered contractile characteristics. Further studies are needed to determine if it can play a role in improving alignment in animal models of strabismus.

Estrogen deficiency induces changes in bone matrix bound water that do not closely correspond with bone turnover

AUTHORS

Corinne E. Metzger, Peter Olayooye, Landon Y. Tak, Oli Culpepper, Alec N. LaPlant, Peter Jalaie, Pearl-Marie Andoh, Wikum Bandara, Olivia N. Reul, Andrew A. Tomaschke, Rachel K. Surowiec

ABSTRACT

Postmenopausal osteoporosis, marked by estrogen deficiency, is a major contributor to osteoporotic fractures, yet early prediction of fractures in this population remains challenging. Our goal was to explore the temporal changes in bone-specific inflammation, oxidative stress, bone turnover, and bone-matrix water, and their relationship with estrogen deficiency-induced modifications in bone structure and mechanical properties. Additionally, we sought to determine if emerging clinically translatable imaging techniques could capture early bone modifications prior to standard clinical imaging.

Two-month-old female Sprague Dawley rats (n = 48) underwent ovariectomy (OVX, n = 24) or sham operations (n = 24). A subgroup of n = 8 rats per group was sacrificed at 2-, 5-, and 10-weeks post-surgery to assess the temporal relationships of inflammation, oxidative stress, bone turnover, bone matrix water, mechanics, and imaging outcomes.

OVX rats exhibited higher body weight compared to sham rats at all time points. By 5-weeks, OVX animals showed elevated markers of inflammation and oxidative stress in cortical bone, which persisted throughout the study, while cortical bone formation rate did not differ from sham until 10-weeks. DXA outcomes did not reveal differences between OVX and sham at any time point. Bound water, assessed using ultrashort echo time magnetic resonance imaging (UTE MRI), was lower in OVX at the earliest time point (2-weeks) and reduced again at 10-weeks with no difference at 5-weeks.

These data demonstrate that bound water assessment using novel UTE MRI technology was lower at the earliest time point following OVX. However, no temporal relationship with bone turnover, inflammation, or oxidative stress was observed at the time points assessed in this study. These findings underscore both the increased need to understand bone hydration changes and highlight the usefulness of UTE MRI for non-invasive bone hydration measurements.

Deficiency of Trps1 in Cementoblasts Impairs Cementogenesis and Tooth Root Formation

AUTHORS

Kaoru Fujikawa, Mairobys Socorro, Lyudmila Lukashova, Priyanka Hoskere, Paulina Keskinidis, Kostas Verdelis, Dobrawa Napierala

ABSTRACT

Cementum is the least studied of all mineralized tissues and little is known about mechanisms regulating its formation. Therefore, the goal of this study was to provide new insights into the transcriptional regulation of cementum formation by determining the consequences of the deficiency of the Trps1 transcription factor in cementoblasts. We used Trps1Col1a1 cKO (2.3Co1a1-CreERT2;Trps1fl/fl) mice, in which Trps1 is deleted in cementoblasts. Micro-computed tomography analyses of molars of 4-week-old males and females demonstrated significantly shorter roots with thinner mineralized tissues (root dentin and cementum) in Trps1Col1a1 cKO compared to WT mice. Semi-quantitative histological analyses revealed a significantly reduced area of cellular cementum and localized deficiencies of acellular cementum in Trps1Col1a1 cKO mice. Immunohistochemical analyses revealed clustering of cementoblasts at the apex of roots, and intermittent absence of cementoblasts on Trps1Col1a1 cKO cementum surfaces. Fewer Osterix-positive cells adjacent to cellular cementum were also detected in Trps1Col1a1 cKO compared to WT mice. Decreased levels of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme required for proper cementogenesis, were apparent in cementum, periodontal ligament, and alveolar bone of Trps1Col1a1 cKO. There were no apparent differences in levels of bone sialoprotein (Bsp) in cementum. Quantitative analyses of picrosirius red-stained periodontal ligament revealed shorter and disorganized collagen fibers in Trps1Col1a1 cKO mice demonstrating impaired periodontal structure. In conclusion, this study has identified Trps1 transcription factor as one of the important regulators of cellular and acellular cementum formation. Furthermore, this study suggests that Trps1 supports the function of cementoblasts by upregulating expression of the major proteins required for cementogenesis, such as Osterix and TNAP.

Epimedin A inhibits the PI3K/AKT/NF-κB signalling axis and osteoclast differentiation by negatively regulating TRAF6 expression

AUTHORS

Jun Li, Jia J. Wei, Cen H. Wu, Tao Zou, Hong Zhao, Tian Q. Huo, Cheng J. Wei, Ting Yang

ABSTRACT

Background

Epimedin A (EA) has been shown to suppress extensive osteoclastogenesis and bone resorption, but the effects of EA remain incompletely understood. The aim of our study was to investigate the effects of EA on osteoclastogenesis and bone resorption to explore the corresponding signalling pathways.

Methods

Rats were randomly assigned to the sham operation or ovariectomy group, and alendronate was used for the positive control group. The therapeutic effect of EA on osteoporosis was systematically analysed by measuring bone mineral density and bone biomechanical properties. In vitro, RAW264.7 cells were treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) to induce osteoclast differentiation. Cell viability assays, tartrate-resistant acid phosphatase (TRAP) staining, and immunofluorescence were used to elucidate the effects of EA on osteoclastogenesis. In addition, the expression of bone differentiation-related proteins or genes was evaluated using Western blot analysis or quantitative polymerase chain reaction (PCR), respectively.

Results

After 3 months of oral EA intervention, ovariectomized rats exhibited increased bone density, relative bone volume, trabecular thickness, and trabecular number, as well as reduced trabecular separation. EA dose-dependently normalized bone density and trabecular microarchitecture in the ovariectomized rats. Additionally, EA inhibited the expression of TRAP and NFATc1 in the ovariectomized rats. Moreover, the in vitro results indicated that EA inhibits osteoclast differentiation by suppressing the TRAF6/PI3K/AKT/NF-κB pathway. Further studies revealed that the effect on osteoclast differentiation, which was originally inhibited by EA, was reversed when the TRAF6 gene was overexpressed.

Conclusions

The findings indicated that EA can negatively regulate osteoclastogenesis by inhibiting the TRAF6/PI3K/AKT/NF-κB axis and that ameliorating ovariectomy-induced osteoporosis in rats with EA may be a promising potential therapeutic strategy for the treatment of osteoporosis.

The Regulation of Hyperglycaemia-Induced Exosomal Mir-6499-3p Derived from Vascular Endothelial Cell on Calcification/Senescence of Vascular Smooth Muscle Cells

AUTHORS

Jiayu Zhong, MingHao Yuan, Shuo Hu, En Zhou

ABSTRACT

Diabetes is a prevalent metabolic condition that is closely linked to aging, and its biggest associated risk is vascular related complications. The calcification and aging of blood vessels in diabetes play a significant role in the development of diabetic vascular complications. Growing evidence links exosomal microRNA to the process of diabetic vascular complications. The present study aims to explore the expression, regulatory mechanisms and functions of exosomal miR-6499-3p in the process of diabetic vascular complications. VSMCs could take up exosomes isolated from HUVEC(ECs-exosome) treated with high glucose(HG). These exosomes induced the calcification and senescence of VSMCs through a paracrine mechanism. We then found that HG elevated the expression level of miR-6499-3p both in HUVECs and ECs-exosome. Calcification and senescence of VSMCs can be promoted by ECs-exosomes following overexpression of miR-6499-3p. Furthermore, we demonstrated that the effects of exosomes on the calcification and senescence of VSMCs is mediated by Sclerosin-containing domain SOSTDC1, the potential target of the miR-6499-3p. Our data has shown that a specific role of exosomes from HG-treated HUVEC in regulating the calcification and senescence of VSMCs in a paracrine manner through the miR-6499-3p/SOSTDC1 pathway. Modulation of exosomal miR-6499-3p may provide a novel perspective on the treatment of diabetic vascular complications.