Zixue Jin

miR-34a Blocks Osteoporosis and Bone Metastasis by Inhibiting Osteoclastogenesis and Tgif2

Bone-resorbing osteoclasts significantly contribute to osteoporosis and bone metastases of cancer. MicroRNAs play important roles in physiology and disease, and present tremendous therapeutic potential. Nonetheless, how microRNAs regulate skeletal biology is underexplored. Here we identify miR-34a as a novel and critical suppressor of osteoclastogenesis, bone resorption and the bone metastatic niche. miR-34a is downregulated during osteoclast differentiation. Osteoclastic miR-34a-overexpressing transgenic mice exhibit lower bone resorption and higher bone mass. Conversely, miR-34a knockout and heterozygous mice exhibit elevated bone resorption and reduced bone mass. Consequently, ovariectomy-induced osteoporosis, as well as bone metastasis of breast and skin cancers, are diminished in osteoclastic miR-34a transgenic mice, and can be effectively attenuated by miR-34a nanoparticle treatment. Mechanistically, we identify transforming growth factor-β-induced factor 2 (Tgif2) as an essential direct miR-34a target that is pro-osteoclastogenic. Tgif2 deletion reduces bone resorption and abolishes miR-34a regulation. Together, using mouse genetic, pharmacological and disease models, we reveal miR-34a as a key osteoclast suppressor and a potential therapeutic strategy to confer skeletal protection and ameliorate bone metastasis of cancers.

Accelerated liver fibrosis in hepatitis B virus transgenic mice: involvement of natural killer T cells.

The immunopathogenic process from hepatitis B virus (HBV) infection to liver fibrosis is incompletely understood because it lacks an animal model. In this study we observed the development of liver fibrosis in HBV transgenic (HBV‐tg) mice and found the roles of natural killer T (NKT) cells in HBV‐related liver fibrosis. We found liver fibrosis spontaneously developed in HBV‐tg mice with the elevated transcription of col1a1, matrix metalloproteinase (MMP)2, and tissue inhibitor of metalloproteinase (TIMP)1. Mice were then injected with repetitive hepatotoxin carbon tetrachloride (CCl4) to induce prominent liver fibrosis. After chronic CCl4 treatment, the serum alanine aminotransferase (ALT) was higher, the liver regenerative nodules became more and bigger, and the fibrosis area was remarkably increased in HBV‐tg mice than in C57BL/6 mice. Moreover, the increase in col1a1 and MMP2 transcription was greater, with a sustaining high level of TIMP1 and a greater activation of hepatic stellate cells (HSCs) in the livers of CCl4‐treated HBV‐tg mice. Our data also showed that there were more liver mononuclear cells (MNCs) in HBV‐tg mice after CCl4 injection, and Rag1−/− mice adoptive transferred lymphocytes from HBV‐tg mice displayed increased collagen deposition. Further study demonstrated the number of liver NKT cells increased after CCl4 treatment and NKT cells were overactivated in HBV‐tg mice in the long term. It was further confirmed that NKT cells were critical for HSCs activation by depletion of NKT cells of HBV‐tg mice and adoptive transfer of purified NKT cells from HBV‐tg mice into recipient Rag1−/− mice. The inflammatory cytokines IL‐4 and IL‐13 produced by NKT cells played a pivotal role in HSCs activation in an in vitro coculture experiment. Conclusion: These data suggest that NKT cells from HBV‐tg mice induce the HSC activation in liver fibrogenesis. (HEPATOLOGY 2011;.)

HDAC9 Inhibits Osteoclastogenesis via Mutual Suppression of PPARγ/RANKL Signaling.

Recent studies suggest that the class II histone deacetylase (HDAC)9 plays important roles in physiology such as metabolism and immunity. Here, we report that HDAC9 also controls bone turnover by suppressing osteoclast differentiation and bone resorption. HDAC9 expression is down-regulated during osteoclastogenesis. Ex vivo osteoclast differentiation is accelerated by HDAC9 deletion but diminished by HDAC9 overexpression. HDAC9 knockout mice exhibit elevated bone resorption and lower bone mass. Bone marrow transplantation reveal that the osteoclastogenic defects are intrinsic to the hematopoietic lineage, because the excessive bone resorption phenotype can be conferred in wild-type (WT) mice receiving HDAC9-null bone marrow, and rescued in HDAC9-null mice receiving WT bone marrow. Mechanistically, HDAC9 forms a negative regulatory loop with peroxisome proliferator-activated receptor gamma (PPARg) and receptor activator of nuclear factor kappa-B ligand (RANKL) signaling. On one hand, PPARγ and nuclear factor κB suppress HDAC9 expression, on the other hand, HDAC9 inhibits PPARγ activity in synergy with silencing mediator of retinoic acid and thyroid hormone receptors (SMRT)/NCoR corepressors. These findings identify HDAC9 as a novel, important and physiologically relevant modulator of bone remodeling and skeletal homeostasis.

HDAC7 Inhibits Osteoclastogenesis by Reversing RANKL-Triggered β-Catenin Switch

The bone-resorbing osteoclast is essential for skeletal remodeling, yet its deregulation contributes to diseases such as osteoporosis and cancer bone metastasis. Here we identify histone deacetylase 7 (HDAC7) as a key negative regulator of osteoclastogenesis and bone resorption using both in vitro cellular and molecular analyses and in vivo characterization of conditional HDAC7-knockout mice. Bone marrow osteoclast differentiation assays reveal that HDAC7 overexpression suppresses, whereas HDAC7 deletion enhances, osteoclastogenesis. Mechanistically, in the absence of receptor activator of nuclear factor κ-B ligand (RANKL), HDAC7 attenuates β-catenin function and cyclin D1 expression, thereby reducing precursor proliferation; upon RANKL activation, HDAC7 suppresses NFATc1 and prevents β-catenin down-regulation, thereby blocking osteoclast differentiation. Consequently, HDAC7 deletion in the osteoclast lineage results in a 26% reduction in bone mass (P = 0.003) owing to 102% elevated bone resorption (P = 0.01). These findings are clinically significant in light of the remarkable therapeutic potentials of HDAC inhibitors for several diseases such as cancer, diabetes, and neurodegeneration.

Minireview: Nuclear Receptor Regulation of Osteoclast and Bone Remodeling

Osteoclasts are bone-resorbing cells essential for skeletal remodeling and regeneration. However, excessive osteoclasts often contribute to prevalent bone degenerative diseases such as osteoporosis, arthritis, and cancer bone metastasis. Osteoclast dysregulation is also associated with rare disorders such as osteopetrosis, pycnodysostosis, Pagets disease, and Gorham-Stout syndrome. The nuclear receptor (NR) family of transcription factors functions as metabolic sensors that control a variety of physiological processes including skeletal homeostasis and serves as attractive therapeutic targets for many diseases. In this review, we highlight recent findings on the new players and the new mechanisms for how NRs regulate osteoclast differentiation and bone resorption. An enhanced understanding of NR functions in osteoclastogenesis will facilitate the development of not only novel osteoprotective medicine but also prudent strategies to minimize the adverse skeletal effects of certain NR-targeting drugs for a better treatment of cancer and metabolic diseases.

Orexin Regulates Bone Remodeling via a Dominant Positive Central Action and a Subordinate Negative Peripheral Action

Orexin neuropeptides promote arousal, appetite, reward, and energy expenditure. However, whether orexin affects bone mass accrual is unknown. Here, we show that orexin functions centrally through orexin receptor 2 (OX2R) in the brain to enhance bone formation. OX2R null mice exhibit low bone mass owing to elevated circulating leptin, whereas central administration of an OX2R-selective agonist augments bone mass. Conversely, orexin also functions peripherally through orexin receptor 1 (OX1R) in the bone to suppress bone formation. OX1R null mice exhibit high bone mass owing to a differentiation shift from marrow adipocyte to osteoblast that results from higher osseous ghrelin expression. The central action is dominant because bone mass is reduced in orexin null and OX1R2R double null mice but enhanced in orexin-overexpressing transgenic mice. These findings reveal orexin as a critical rheostat of skeletal homeostasis that exerts a yin-yang dual regulation and highlight orexin as a therapeutic target for osteoporosis.

Maternal Adiponectin Controls Milk Composition to Prevent Neonatal Inflammation

Adiponectin is an important adipokine. Increasing evidence suggests that altered adiponectin levels are linked with metabolic and inflammatory disorders. Here we report an important yet previously unrecognized function of adiponectin in lactation by which maternal adiponectin determines the inflammatory status in the nursing neonates. Surprisingly, both maternal adiponectin overexpression in the transgenic mice and maternal adiponectin deletion in the knockout mice lead to systemic inflammation in the pups, manifested as transient hair loss. However, distinct mechanisms are involved. Adiponectin deficiency triggers leukocyte infiltration and production of inflammatory cytokines in the lactating mammary gland. In contrast, adiponectin overabundance increases lipid accumulation in the lactating mammary gland, resulting in excessive long-chain saturated fatty acids in milk. Interestingly, in both cases, the inflammation and alopecia in the pups can be rescued by Toll-like receptor (TLR)-2/4 deletion because TLR2/4 double-knockout pups are resistant. Mechanistically, long-chain saturated fatty acid activation of inflammatory genes is TLR2/4 dependent and can be potentiated by proinflammatory cytokines, indicating that the inflammatory stimuli in both scenarios functionally converge by activating the TLR2/4 signaling. Therefore, our findings reveal adiponectin as a dosage-dependent regulator of lactation homeostasis and milk quality that critically controls inflammation in the nursing neonates. Furthermore, these results suggest that inflammatory infantile disorders may result from maternal adiponectin dysregulation that can be treated by TLR2/4 inhibition.

Abstract P6-17-01: MicroRNA-34a suppresses breast cancer bone metastasis by inhibiting osteoclastogenesis and targeting tgif2

Introduction: Approximately 70% of people living with metastatic breast cancer have metastases to their bones. Osteolytic bone metastasis is a common, debilitating and essentially incurable skeletal complication of breast cancer, in which tumor cells migrate to and destroy bones, causing extreme pain, fractures, life-threatening hypercalcemia, limited mobility and eventually motality. The bone resorbing osteoclasts significantly contribute to this process. MicroRNAs (miRNAs) play important roles in physiology and disease, and present tremendous therapeutic potential. Nonetheless, how miRNAs regulate skeletal biology is underexplored. We hypothesize that miRNAs that can suppress osteoclast function may ameliorate breast cancer bone metastasis. Methods: A strategy of ex vivo osteoclast differentiation from mouse bone marrow cells was used to examine the levels of several cancer-related miRNAs during a time course of osteoclastogenesis, and test the effects of miR-34a mimic or inhibitor. Micro-Computed Tomography was conducted to quantify bone mass. ELISA analyses were performed to measure serum bone resorption and bone formation markers. Ovariectomy was employed as a model for postmenopausal osteoporosis. As a model for bone metastases, luciferase labelled bone-metastasis prone MDA-MB-231 human breast cancer cell subline were injected into the left cardiac ventrical of nu/nu mice. Bone metastases were detected and quantified weekly post injection by bioluminescence imaging. Results: MiR-34a is down-regulated during osteoclast differentiation. Osteoclastic miR-34a over-expressing transgenic mice exhibit lower bone resorption and higher bone mass. Conversely, miR-34a knockout and heterozygous mice exhibit elevated bone resorption and reduced bone mass. Consequently, ovariectomy-induced osteoporosis, as well as bone metastasis of breast cancer are diminished in osteoclastic miR-34a transgenic mice. Pharmacologically, systemic delivery of miR-34a mimics via a chitosan nanoparticle vehicle (miR-34a-CH) can target multiple tissues. Our bio-distribution analysis shows that miR-34a-CH delivery to the bone marrow is among the highest compared to other tissues. Administration of miR-34a nanoparticles not only diminished ovariectomy induced bone loss but also attenuated breast cancer bone metastasis. Systemic miR-34a-CH delivery affected neither tumor growth when cancer cells were injected subcutaneously nor tumor metastasis to other organs such as lung when cancer cells were injected intravenously. These results further support the notion that the suppression of bone metastases by miR-34a-CH was mediated by its inhibition of the bone metastatic niche rather than the cancer cells. Mechanistically, we identify the homeodomain transcription factor Tgif2 as an essential direct miR-34a target that is pro-osteoclastogenic. Tgif2 deletion reduces bone resorption and abolishes miR-34a regulation. Conclusion: Using mouse genetic, pharmacological and disease models, we have identified mir-34a as a novel and critical suppressor of osteoclastogenesis, bone resorption and the bone metastatic niche, revealing miR-34a as a potential new therapeutic strategy to confer skeletal protection and ameliorate bone metastasis of breast cancer. Citation Format: Jing Y Krzeszinski, Wei Wei, HoangDinh Huynh, Zixue Jin, Xunde Wang, Tsung-Cheng Chang, Xian-jin Xie, Lin He, Lingegowda S Mangala, Gabriel Lopez-Berestein, Anil K Sood, Joshua T Mendell, Yihong Wan. MicroRNA-34a suppresses breast cancer bone metastasis by inhibiting osteoclastogenesis and targeting tgif2 [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-17-01.