No abstract

Glucocorticoid (GC) therapy is the most frequent cause of secondary osteoporosis. In this study we have demonstrated that GC treatment induced the development of autophagy, preserving osteocyte viability. GC treatment resulted in an increase in autophagy markers and the accumulation of autophagosome vacuoles in vitro and in vivo promoted the onset of the osteocyte autophagy, as determined by expression of autophagy markers in an animal model of GC-induced osteoporosis. An autophagy inhibitor reversed the protective effects of GCs. The effects of GCs on osteocytes were in contrast to tumor necrosis factor α (TNF-α), which induced apoptosis but not autophagy. Together this study reveals a novel mechanism for the effect of GC on osteocytes, shedding new insight into mechanisms responsible for bone loss in patients receiving GC therapy. © 2010 American Society for Bone and Mineral Research.

In response to cellular insult, several pathways can be activated, including necrosis, apoptosis, and autophagy. Because glucocorticoids (GCs) have been shown to induce both osteocyte apoptosis and autophagy, we sought to determine whether osteocyte cell fate in the presence of GCs was dose dependent by performing in vivo and in vitro studies. Male Swiss-Webster mice were treated with slow-release prednisolone pellets at 1.4, 2.8, and 5.6 mg/kg/d for 28 d. An osteocyte cell line, MLO-Y4 cells, was treated with various doses of dexamethasone. We found that GC treatments dose dependently decreased activation of antioxidant-, autophagy-, and antiapoptosis-focused RT-PCR gene pathways in mouse cortical bone. The activation of antioxidant genes was correlated with autophagy gene expression after the GC treatments. The presence of osteocyte autophagy, as detected by immunostaining for LC3, increased ∼50% at the distal femur cortical bone region but not at trabecular bone region at the 1.4 and 2.8 mg/kg/d GC dose levels. The number of apoptotic osteocytes was increased at the cortical bone region by ∼40% initially observed at the 2.8 mg/kg/d dose level. In addition, the presence of the osteocyte autophagy was associated with an increased protein level of cathepsin K in vitro after the GC treatments. In summary, we found that GC treatment dose-dependently decreased antioxidant gene expression, with lower GC doses activating autophagy, whereas a higher dose increased apoptosis. These data suggest that autophagy may provide a mechanism for osteocytes to survive the stress after GC exposure and provide further insight into how GCs alter bone cell fate.

Thiol reactive cyclopentenone prostaglandin, 15-deoxy-Δ12, 14-Prostaglandin J2, induced a novel, non-apoptotic and Map1 LC3 dependent but non-autophagic form of cell death in colon, breast and prostate cancer cell lines, characterized by extensive cytoplasmic vacuolation with dilatation of endoplasmic reticulum. Disruption of sulfhydryl homeostasis, which resulted in ER stress, accumulation of ubiquitinated proteins and subsequent ER dilation, contributed to PPARγ independent cell death by 15d-PGJ2. Absence of intracellular organelles in these vacuoles, shown by Electron Microscopy and unique fragmentation of Lamin B, suggested this form of cell death to be different from autophagy and apoptosis. Cell death induced by 15d-PGJ2 is prevented by cycloheximide and actinomycin D, suggesting a requirement of new protein synthesis for death with cytoplasmic vacuolation. Here, we report for the first time that upregulation and processing of autophagy marker LC3 is an important event in non-autophagic cytoplasmic vacuolation and cell death. Notably, knockdown of LC3 conferred significant protection against 15d-PGJ2 induced cytoplasmic vacuolation and cell death suggesting a novel role of LC3 in a death process other than autophagy.

Gap junctional intercellular communication (GJIC) mediated by connexins, in particular connexin 43 (Cx43), plays important roles in regulating signal transmission among different bone cells and thereby regulates development, differentiation, modeling and remodeling of the bone. GJIC regulates osteoblast formation, differentiation, survival and apoptosis. Osteoclast formation and resorptive ability are also reported to be modulated by GJIC. Furthermore, osteocytes utilize GJIC to coordinate bone remodeling in response to anabolic factors and mechanical loading. Apart from gap junctions, connexins also form hemichannels, which are localized on the cell surface and function independently of the gap junction channels. Both these channels mediate the transfer of molecules smaller than 1.2 kDa including small ions, metabolites, ATP, prostaglandin and IP3. The biological importance of the communication mediated by connexin-forming channels in bone development is revealed by the low bone mass and osteoblast dysfunction in the Cx43-null mice and the skeletal malformations observed in occulodentodigital dysplasia (ODDD) caused by mutations in the Cx43 gene. The current review summarizes the role of gap junctions and hemichannels in regulating signaling, function and development of bone cells.

The increased osteocyte death by oxidative stress (OS) during aging is a major cause contributing to the impairment of bone quality and bone loss. However, the underlying molecular mechanism is largely unknown. Here, we showed that H2O2 induced cell death of primary osteocytes and osteocytic MLO-Y4 cells, and also caused dose-dependent decrease expression of gap junction and hemichannel-forming connexin 43 (Cx43). The decrease of Cx43 expression was also demonstrated with the treatment of other oxidants, rotenone and menadione. Antioxidant reversed the effects of oxidants on Cx43 expression and osteocyte cell death. Cx43 protein was also much lower in the osteocytes from 20-month as opposed to the 5-week or 20-week old mice. Dye transfer assay showed that H2O2 reduced the gap junction intercellular communication (GJIC). In contrast to the effect on GJIC, there was a dose-dependent increase of hemichannel function by H2O2, which was correlated with the increased cell surface expression of Cx43. Cx43 (E2) antibody, an antibody which specifically blocks Cx43 hemichannel activity but not gap junctions completely blocked dye uptake induced by H2O2 and further exacerbated H2O2-induced osteocytic cell death. In addition, knockdown of Cx43 expression by siRNA increased the susceptibility of the cells to OS-induced death. Together, our study provides a novel cell protective mechanism mediated by osteocytic Cx43 channels against OS.

Although the skeleton is one of predominant sites for breast cancer metastasis, why breast cancer cells often become dormant after homing to bone is not well understood. Here, we reported an intrinsic self-defense mechanism of bone cells against breast cancer cells: a critical role of connexin (Cx) 43 hemichannels in osteocytes in the suppression of breast cancer bone metastasis. Cx43 hemichannels allow passage of small molecules between the intracellular and extracellular environments. The treatment of bisphosphonate drugs, either alendronate (ALN) or zoledronic acid (ZOL), opened Cx43 hemichannels in osteocytes. Conditioned media (CM) collected from MLO-Y4 osteocyte cells treated with bisphosphonates inhibited the anchorage-independent growth, migration and invasion of MDA-MB-231 human breast cancer cells and Py8119 mouse mammary carcinoma cells and this inhibitory effect was attenuated with Cx43(E2), a specific hemichannel blocking antibody. The opening of osteocytic Cx43 hemichannels by mechanical stimulation had similar inhibitory effects on breast cancer cells and this inhibition was attenuated by Cx43(E2) antibody as well. These inhibitory effects on cancer cells were mediated by ATP released from osteocyte Cx43 hemichannels. Furthermore, both Cx43 osteocyte-specific knockout mice and osteocyte-specific Δ130–136 transgenic mice with impaired Cx43 gap junctions and hemichannels showed significantly increased tumor growth and attenuated the inhibitory effect of ZOL. However, R76W transgenic mice with functional hemichannels but not gap junctions in osteocytes did not display a significant difference. Together, our studies establish the specific inhibitory role of osteocytic Cx43 hemichannels, and exploiting the activity of this channel could serve as a de novo therapeutic strategy.

Many tumor cells express globally reduced levels of microRNAs (miRNA), suggesting that decreased miRNA expression in premalignant cells contributes to their tumorigenic phenotype. In support of this, Dicer, an RNase III-like enzyme that controls the maturation of miRNA, was recently shown to function as a haploinsufficient tumor suppressor in nonhematopoietic cells. Because the Myc oncoprotein, a critical inducer of B-cell lymphomas, was reported to suppress the expression of multiple miRNAs in lymphoma cells, it was presumed that a deficiency of Dicer and subsequent loss of miRNA maturation would accelerate Myc-induced lymphoma development. We report here that, surprisingly, a haploinsufficiency of Dicer in B cells failed to promote B-cell malignancy or accelerate Myc-induced B-cell lymphomagenesis in mice. Moreover, deletion of Dicer in B cells of CD19-cre + /Eμ-myc mice significantly inhibited lymphomagenesis, and all lymphomas that did arise in these mice lacked functional Cre expression and retained at least one functional Dicer allele. Uncharacteristically, the lymphomas that frequently developed in the CD19-cre + /Dicer fl/fl /Eμ-myc mice were of very early precursor B-cell origin, a stage of B-cell development prior to Cre expression. Therefore, loss of Dicer function was not advantageous for lymphomagenesis, but rather, Dicer ablation was strongly selected against during Myc-induced B-cell lymphoma development. Moreover, deletion of Dicer in established B-cell lymphomas resulted in apoptosis, revealing that Dicer is required for B-cell lymphoma survival. Thus, Dicer does not function as a haploinsufficient tumor suppressor in B cells and is required for B-cell lymphoma development and survival. Cancer Res; 70(14); 6083-92. ©2010 AACR.