Date Published: April 10, 2019
Publisher: Public Library of Science
Author(s): Priyatha Premnath, Leah Ferrie, Dante Louie, Steven Boyd, Roman Krawetz, Dominique Heymann.
p21(WAF1/CIP1/SDI1) is a critical sentinel of the cell cycle that plays an important role in determining cell fate with respect to proliferation, differentiation and apoptosis. Recent studies have demonstrated that inhibition/loss of p21 promotes osteo-chondro differentiation in progenitor/stem cells, and that p21 knockout (p21-/-) mice demonstrate enhanced bone regeneration compared to wild-type controls after a non-critical size defect. It was therefore hypothesized that the absence of p21 may also protect against bone loss through enhancing bone formation, tilting the balance away from bone resorption, in an ovariectomy-induced osteopenia mouse model, investigated via microCT imaging. While p21-/- mice demonstrated significantly less bone loss after ovariectomy compared to wild-type controls, no increase in the number osteoclasts or osteoblasts in the bone or bone marrow was observed, nor was there an increase in osteoclast activity. Therefore, while the absence of p21 protected mice against estrogen mediated bone loss, the mechanisms/pathways responsible remained elusive. This study demonstrates that p21 may play a significant role in bone remodeling, and a better understanding of how the p21 pathway regulates bone anabolism and catabolism could lead to novel therapies for osteoporosis in the future.
Osteoporosis is characterized by the deterioration of bone architecture and consequent loss in bone strength, and its onset is based on age and/or menopause. In menopause, a deficiency in estrogen exacerbates osteoclast resorption compared to matrix being laid down by osteoblasts by elevating cytokines such as tumor necrosis factor α(TNF-α), interleukin-1(IL-1), IL-6 and interferon γ (IFN-γ) . Current therapies for osteoporosis directly target osteoclasts and impede resorption via pharmacological anti-resorptive drugs such as bisphosphonates. Other strategies include treatment with selective estrogen receptor modulators or receptor activator of nuclear factor kappa B ligand inhibitors, but all have considerable disadvantages [1,2]. The primary drawback with most current treatment options is that while they prevent further resorption of bone, they do not promote the regeneration of bone that has already been lost to the disease process, leaving the bone in a weakened state.
p21cip1/waf1 is regulated by the tumor suppressor gene p53 and plays an important role in cell cycle regulation. Inhibition of p21 has been implicated in regeneration of tissues such as liver , and more recently bone regeneration after fracture . The exact mechanism behind p21 regulated regeneration remains elusive despite studies suggesting that enhanced proliferation of cells may be a contributing factor. The results from the current study demonstrate that p21-/- mice are protected against bone loss post-OVX, while C57BL/6 mice show significant bone loss in spite of similar cell numbers in the bone marrow (MSCs, osteoblasts and osteoclasts). Since inhibition of p21 in chondrocytes induces their apoptosis and therefore could influence skeletal development , it is possible that differences in endochondral ossification between these strains may contribute to the findings. However, when the thickness of growth plate was assessed, similar to a study by Gerber et al. , no significant differences between the two mice strains was observed (Figure G in S1 File). These results suggest that the mechanism by which p21-/- mice retain/regenerate bone post-OVX is not likely based on osteoclast vs. osteoblast function, localization and/or number, and therefore may be a result of a ‘short circuit’ in the OVX disease mechanism itself. While the exact cellular mechanism by which estrogen controls metabolic homeostasis and bone resorption is not fully known, it is well established that diminished estrogen levels through OVX surgery has a dramatic and immediate effect on bone homeostasis in mice through which is mediated through an increase in osteoclast numbers and activity . Interestingly, a direct link has been demonstrated between p21 and estrogen regulation, with previous studies showing that estrogen is a potent inducer of p21 expression . Furthermore, p21 can regulate the expression of the estrogen receptor , and can therefore act as a potent inhibitor of the estrogen signaling cascade . Hypothetically then, if the pathway by which estrogen regulates bone homeostasis is interrupted by OVX (e.g. decreased estrogen levels) and p21 is a negative regulator of estrogen signaling, then a double knock-down/knockout of estrogen and p21 respectively, may result in the expected bone loss phenotype being absent. While this hypothesis has yet to be tested, it is supported by our previous study of the role of p21 in bone regeneration, since p21-/- mice demonstrated enhanced bone regeneration after a burr-hole injury compared to C57BL/6 mice, yet no difference in osteoblast vs. osteoclast function was observed . If p21 is downstream and inhibits estrogen signaling, then it may be possible for a p21-/- mice on an estrogen competent background to show the opposite effect vs. when estrogen is removed (e.g. favoring bone formation vs. resorption).
Our findings demonstrate that p21-/- mice are protected against osteopenia post-OVX compared to C57BL/6 mice. These results suggest a larger role of p21 in bone homeostasis and potentially points to a direct relationship between p21 and estrogen. A better understanding of the mechanism behind estrogen and p21 related bone formation may result in novel therapies for bone repair and/or osteoporosis.