LIS1 Regulates Osteoclast Formation and Function through Its Interactions with Dynein/Dynactin and Plekhm1

Microtubule organization and lysosomal secretion are both critical for the activation and function of osteoclasts, highly specialized polykaryons that are responsible for bone resorption and skeletal homeostasis. Here, we have identified a novel interaction between microtubule regulator LIS1 and Plekhm1, a lysosome-associated protein implicated in osteoclast secretion. Decreasing LIS1 expression by shRNA dramatically attenuated osteoclast formation and function, as shown by a decreased number of mature osteoclasts differentiated from bone marrow macrophages, diminished resorption pits formation, and reduced level of CTx-I, a bone resorption marker. The ablated osteoclast formation in LIS1-depleted macrophages was associated with a significant decrease in macrophage proliferation, osteoclast survival and differentiation, which were caused by reduced activation of ERK and AKT by M-CSF, prolonged RANKL-induced JNK activation and declined expression of NFAT-c1, a master transcription factor of osteoclast differentiation. Consistent with its critical role in microtubule organization and dynein function in other cell types, we found that LIS1 binds to and colocalizes with dynein in osteoclasts. Loss of LIS1 led to disorganized microtubules and aberrant dynein function. More importantly, the depletion of LIS1 in osteoclasts inhibited the secretion of Cathepsin K, a crucial lysosomal hydrolase for bone degradation, and reduced the motility of osteoclast precursors. These results indicate that LIS1 is a previously unrecognized regulator of osteoclast formation, microtubule organization, and lysosomal secretion by virtue of its ability to modulate dynein function and Plekhm1

Recruitment, augmentation and apoptosis of rat osteoclasts in 1,25-(OH)2D3 response to short-term treatment with 1,25-dihydroxyvitamin D3in vivo

Background Although much is known about the regulation of osteoclast (OC) formation and activity, little is known about OC senescence. In particular, the fate of of OC seen after 1,25-(OH)2D3 administration in vivo is unclear. There is evidence that the normal fate of OC is to undergo apoptosis (programmed cell death). We have investigated the effect of short-term application of high dose 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on OC apoptosis in an experimental rat model. Methods OC recruitment, augmentation and apoptosis was visualised and quantitated by staining histochemically for tartrate resistant acid phosphatase (TRAP), double staining for TRAP/ED1 or TRAP/DAPI, in situ DNA fragmentation end labelling and histomorphometric analysis. Results Short-term treatment with high-dose 1,25-(OH)2D3 increased the recruitment of OC precursors in the bone marrow resulting in a short-lived increase in OC numbers. This was rapidly followed by an increase in the number of apoptotic OC and their subsequent removal. The response of OC to 1,25-(OH)2D3 treatment was dose and site dependent; higher doses producing stronger, more rapid responses and the response in the tibiae being consistently stronger and more rapid than in the vertebrae. Conclusions This study demonstrates that (1) after recruitment, OC are removed from the resorption site by apoptosis (2) the combined use of TRAP and ED1 can be used to identify OC and their precursors in vivo (3) double staining for TRAP and DAPI or in situ DNA fragmentation end labelling can be used to identify apoptotic OC in vivo

Estrogens Attenuate Oxidative Stress and the Differentiation and Apoptosis of Osteoblasts by DNA-Binding-Independent Actions of the ERα

Estrogens diminish oxidative stress in bone and bone marrow, attenuate the generation of osteoblasts, and decrease the prevalence of mature osteoblast apoptosis. We have searched for the molecular mechanism of these effects using as tools a mouse model bearing an estrogen receptor α (ERα) knock-in mutation that prevents binding to DNA (ERαNERKI/−) and several osteoblast progenitor cell models expressing the wild-type ERα or the ERαNERKI/−. We report that the ability of estrogens to diminish the generation of reactive oxygen species, stimulate the activity of glutathione reductase, and decrease the phosphorylation of p66shc, as well as osteoblastogenesis and osteoblast number and apoptosis, were fully preserved in ERαNERKI/− mice, indicating that the DNA-binding function of the ERα is dispensable for all these effects. Consistent with the attenuation of osteoblastogenesis in this animal model, 17β-estradiol attenuated bone morphogenetic protein 2 (BMP-2)–induced gene transcription and osteoblast commitment and differentiation in murine and human osteoblastic cell lines. Moreover, 17β-estradiol attenuated BMP-2-induced differentiation of primary cultures of calvaria- or bone marrow–derived osteoblastic cells from ERαNERKI/− mice as effectively as in cells from wild-type littermates. The inhibitory effect of the hormone on BMP-2 signaling resulted from an ERα-mediated activation of ERKs and the phosphorylation of Smad1 at the linker region of the protein, which leads to proteasomal degradation. These results illustrate that the effects of estrogens on oxidative stress and the birth and death of osteoblasts do not require the binding of ERα to DNA response elements, but instead they result from the activation of cytoplasmic kinases. © 2010 American Society for Bone and Mineral Researc

Conditional deletion of hypothalamic Y2 receptors reverts gonadectomy-induced bone loss in adult mice

Reduction in levels of sex hormones at menopause in women is associated with two common, major outcomes, the accumulation of white adipose tissue, and the progressive loss of bone because of excess osteoclastic bone resorption exceeding osteoblastic bone formation. Current antiresorptive therapies can reduce osteoclastic activity but have only limited capacity to stimulate osteoblastic bone formation and restore lost skeletal mass. Likewise, the availability of effective pharmacological weight loss treatments is currently limited. Here we demonstrate that conditional deletion of hypothalamic neuropeptide Y2 receptors can prevent ongoing bone loss in sex hormone-deficient adult male and female mice. This benefit is attributable solely to activation of an anabolic osteoblastic bone formation response that counterbalances persistent elevation of bone resorption, suggesting the Y2-mediated anabolic pathway to be independent of sex hormones. Furthermore, the increase in fat mass that typically occurs after ovariectomy is prevented by germ line deletion of Y2 receptors, whereas in male mice body weight and fat mass were consistently lower than wild-type regardless of sex hormone status. Therefore, this study indicates a role for Y2 receptors in the accumulation of adipose tissue in the hypogonadal state and demonstrates that hypothalamic Y2 receptors constitutively restrain osteoblastic activity even in the absence of sex hormones. The increase in bone formation after release of this tonic inhibition suggests a promising new avenue for osteoporosis treatment

Reduced Reactivation from Dormancy but Maintained Lineage Choice of Human Mesenchymal Stem Cells with Donor Age

Mesenchymal stem cells (MSC) are promising for cell-based regeneration therapies but up to date it is still controversial whether their function is maintained throughout ageing. Aim of this study was to address whether frequency, activation in vitro, replicative function, and in vitro lineage choice of MSC is maintained throughout ageing to answer the question whether MSC-based regeneration strategies should be restricted to younger individuals. MSC from bone marrow aspirates of 28 donors (5–80 years) were characterized regarding colony-forming unit-fibroblast (CFU-F) numbers, single cell cloning efficiency (SSCE), osteogenic, adipogenic and chondrogenic differentiation capacity in vitro. Alkaline phosphatase (ALP) activity, mineralization, Oil Red O content, proteoglycan- and collagen type II deposition were quantified. While CFU-F frequency was maintained, SSCE and early proliferation rate decreased significantly with advanced donor age. MSC with higher proliferation rate before start of induction showed stronger osteogenic, adipogenic and chondrogenic differentiation. MSC with high osteogenic capacity underwent better chondrogenesis and showed a trend to better adipogenesis. Lineage choice was, however, unaltered with age. Conclusion: Ageing influenced activation from dormancy and replicative function of MSC in a way that it may be more demanding to mobilize MSC to fast cell growth at advanced age. Since fast proliferation came along with high multilineage capacity, the proliferation status of expanded MSC rather than donor age may provide an argument to restrict MSC-based therapies to certain individuals

PTHrP Induces Autocrine/Paracrine Proliferation of Bone Tumor Cells through Inhibition of Apoptosis

Giant Cell Tumor of Bone (GCT) is an aggressive skeletal tumor characterized by local bone destruction, high recurrence rates and metastatic potential. Previous work in our lab has shown that the neoplastic cell of GCT is a proliferating pre-osteoblastic stromal cell in which the transcription factor Runx2 plays a role in regulating protein expression. One of the proteins expressed by these cells is parathryroid hormone-related protein (PTHrP). The objectives of this study were to determine the role played by PTHrP in GCT of bone with a focus on cell proliferation and apoptosis. Primary stromal cell cultures from 5 patients with GCT of bone and one lung metastsis were used for cell-based experiments. Control cell lines included a renal cell carcinoma (RCC) cell line and a human fetal osteoblast cell line. Cells were exposed to optimized concentrations of a PTHrP neutralizing antibody and were analyzed with the use of cell proliferation and apoptosis assays including mitochondrial dehydrogenase assays, crystal violet assays, APO-1 ELISAs, caspase activity assays, flow cytometry and immunofluorescent immunohistochemistry. Neutralization of PTHrP in the cell environment inhibited cell proliferation in a consistent manner and induced apoptosis in the GCT stromal cells, with the exception of those obtained from a lung metastasis. Cell cycle progression was not significantly affected by PTHrP neutralization. These findings indicate that PTHrP plays an autocrine/paracrine neoplastic role in GCT by allowing the proliferating stromal cells to evade apoptosis, possibly through non-traditional caspase-independent pathways. Thus PTHrP neutralizing immunotherapy is an intriguing potential therapeutic strategy for this tumor