Knockdown of formin mDia2 alters lamin B1 levels and increases osteogenesis in stem cells.

Nuclear actin plays a critical role in mediating mesenchymal stem cell (MSC) fate commitment. In marrow-derived MSCs, the principal diaphanous-related formin Diaph3 (mDia2) is present in the nucleus and regulates intranuclear actin polymerization, whereas Diaph1 (mDia1) is localized to the cytoplasm and controls cytoplasmic actin polymerization. We here show that mDia2 can be used as a tool to query actin-lamin nucleoskeletal structure. Silencing mDia2 affected the nucleoskeletal lamin scaffold, altering nuclear morphology without affecting cytoplasmic actin cytoskeleton, and promoted MSC differentiation. Attempting to target intranuclear actin polymerization by silencing mDia2 led to a profound loss in lamin B1 nuclear envelope structure and integrity, increased nuclear height, and reduced nuclear stiffness without compensatory changes in other actin nucleation factors. Loss of mDia2 with the associated loss in lamin B1 promoted Runx2 transcription and robust osteogenic differentiation and suppressed adipogenic differentiation. Hence, mDia2 is a potent tool to query intranuclear actin-lamin nucleoskeletal structure, and its presence serves to retain multipotent stromal cells in an undifferentiated state.

Differences in bone structure and unloading-induced bone loss between C57BL/6N and C57BL/6J mice.

The C57BL/6 mouse, the most frequently utilized animal model in biomedical research, is in use as several substrains, all of which differ by a small array of genomic differences. Two of these substrains, C57BL/6J (B6J) and C57BL/6N (B6N), are commonly used but it is unclear how phenotypically similar or different they are. Here, we tested whether adolescent B6N mice have a bone phenotype and respond to the loss of weightbearing differently than B6J. At 9 weeks of age, normally ambulating B6N had lower trabecular bone volume fraction but greater bone formation rates and osteoblast surfaces than corresponding B6J. At 11 weeks of age, differences in trabecular indices persisted between the substrains but differences in cellular activity had ceased. Cortical bone indices were largely similar between the two substrains. Hindlimb unloading (HLU) induced similar degeneration of trabecular architecture and cellular activity in both substrains when comparing 11-week-old HLU mice to 11-week-old controls. However, unloaded B6N mice had smaller cortices than B6J. When comparing HLU to 9 weeks baseline control mice, deterioration in trabecular separation, osteoblast indices, and endocortical variables was significantly greater in B6N than B6J. These data indicate specific developmental differences in bone formation and morphology between B6N and B6J mice, giving rise to a differential response to mechanical unloading that may be modulated, in part, by the genes Herc2, Myo18b, and Acan. Our results emphasize that these substrains cannot be used interchangeably at least for investigations in which the phenotypic makeup and its response to extraneous stimuli are of interest.

Modulation of unloading-induced bone loss in mice with altered ERK signaling.

Genetic variations mediate skeletal responsiveness to mechanical unloading, with individual space travelers exhibiting large variations in the extent of bone loss. We previously identified genomic regions harboring several hundred genes that can modulate the magnitude of skeletal adaptation to mechanical unloading. Here, bioinformatic filters aided in shortlisting 30 genes with bone-related and mechanoregulatory roles. The genes CD44, FGF2, NOD2, and Fas, all associated with ERK signaling, were then functionally tested in hindlimb-unloaded (HLU) knockout (KO) mice. Compared to their respective normally ambulating wildtype (WT) controls, all KO strains, except Fas mice, had lower trabecular bone volume, bone volume fraction, and/or trabecular number. For cortical bone and compared to ambulatory WT mice, CD44(-/-) had impaired properties while FGF2(-/-) showed enhanced indices. NOD2(-/-) and Fas(-/-) did not have a cortical phenotype. In all KO and WT groups, HLU resulted in impaired trabecular and cortical indices, primarily due to trabecular tissue loss and mitigation of cortical bone growth. The difference in trabecular separation between HLU and ambulatory controls was significantly greater in CD44(-/-) and NOD2(-/-) mice than in WT mice. In cortical bone, differences in cortical thickness, total pore volume, and cortical porosity between HLU and controls were aggravated in CD44(-/-) mice. In contrast, deletion of NOD2 and Fas genes mitigated the differences in Po.V between HLU and control mice. Together, we narrowed a previous list of QTL-derived candidate genes from over 300 to 30, and showed that CD44, NOD2, and Fas have distinct functions in regulating changes in trabecular and cortical bone indices during unloading.

Exercise Degrades Bone in Caloric Restriction, Despite Suppression of Marrow Adipose Tissue (MAT).

Marrow adipose tissue (MAT) and its relevance to skeletal health during caloric restriction (CR) is unknown: It remains unclear whether exercise, which is anabolic to bone in a calorie-replete state, alters bone or MAT in CR. We hypothesized that response of bone and MAT to exercise in CR differs from the calorie-replete state. Ten-week-old female B6 mice fed a regular diet (RD) or 30% CR diet were allocated to sedentary (RD, CR, n = 10/group) or running exercise (RD-E, CR-E, n = 7/group). After 6 weeks, CR mice weighed 20% less than RD, p < 0.001; exercise did not affect weight. Femoral bone volume (BV) via 3D MRI was 20% lower in CR versus RD (p < 0.0001). CR was associated with decreased bone by µCT: Tb.Th was 16% less in CR versus RD, p < 0.003, Ct.Th was 5% less, p < 0.07. In CR-E, Tb.Th was 40% less than RD-E, p < 0.0001. Exercise increased Tb.Th in RD (+23% RD-E versus RD, p < 0.003) but failed to do so in CR. Cortical porosity increased after exercise in CR (+28%, p = 0.04), suggesting exercise during CR is deleterious to bone. In terms of bone fat, metaphyseal MAT/ BV rose 159% in CR versus RD, p = 0.003 via 3D MRI. Exercise decreased MAT/BV by 52% in RD, p < 0.05, and also suppressed MAT in CR (-121%, p = 0.047). Histomorphometric analysis of adipocyte area correlated with MAT by MRI (R2 = 0.6233, p < 0.0001). With respect to bone, TRAP and Sost mRNA were reduced in CR. Intriguingly, the repressed Sost in CR rose with exercise and may underlie the failure of CR-bone quantity to increase in response to exercise. Notably, CD36, a marker of fatty acid uptake, rose 4088% in CR (p < 0.01 versus RD), suggesting that basal increases in MAT during calorie restriction serve to supply local energy needs and are depleted during exercise with a negative impact on bone. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Absence of complement factor H alters bone architecture and dynamics.

Complement system is an important arm of the immune system that promotes inflammation. Complement Factor H (FH) is a critical regulator of the alternative complement pathway. Its absence causes pathology in different organs resulting in diseases such as age related macular degeneration and dense deposit disease. Recent studies suggest that the complement system plays a role in bone development and homeostasis. To determine the role of FH in bone architecture, we studied the FH knockout (fh-/-) mice. 3D reconstructions of femur from 16 week old fh-/- mice reveal significant changes, such as decreased BV/TV (4.5%, p < 0.02), trabecular number (22%, p < 0.01), tissue mineral density (16%, p < 0.04), and increased marrow area (16% p < 0.01), compared to their wild type (WT) counterparts. Kidney function and histology remained normal indicating that bone changes occurred prior to kidney dysfunction. Next we examined cultured osteoblasts and osteoclasts isolated from bone marrow. FH is expressed ubiquitously in the osteoblasts and in the cytoplasm of osteoclasts. The changes caused by absence of FH include: increase in number of osteoblasts (362%) and osteoclasts (342%), increase in RNA (180%) and protein expression of cathepsin K and increased osteoclast function (pit formation, 233%). Actin rearrangement in both osteoblasts and osteoclasts was altered, with a loss of integrity of the F-actin ring at the periphery of the osteoclasts. For the first time our studies demonstrate a direct role of FH in the maintenance of bone structure and function and is highlighted as a promising therapeutic target in bone diseases.