Rekha Kar

Glucocorticoid-induced autophagy in osteocytes.

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.

Biological Role of Connexin Intercellular Channels and Hemichannels

Gap junctions (GJ) and hemichannels (HC) formed from the protein subunits called connexins are transmembrane conduits for the exchange of small molecules and ions. Connexins and another group of HC-forming proteins, pannexins comprise the two families of transmembrane proteins ubiquitously distributed in vertebrates. Most cell types express more than one connexin or pannexin. While connexin expression and channel activity may vary as a function of physiological and pathological states of the cell and tissue, only a few studies suggest the involvement of pannexin HC in acquired pathological conditions. Importantly, genetic mutations in connexin appear to interfere with GJ and HC function which results in several diseases. Thus connexins could serve as potential drug target for therapeutic intervention. Growing evidence suggests that diseases resulting from HC dysfunction might open a new direction for development of specific HC reagents. This review provides a comprehensive overview of the current studies of GJ and HC formed by connexins and pannexins in various tissue and organ systems including heart, central nervous system, kidney, mammary glands, ovary, testis, lens, retina, inner ear, bone, cartilage, lung and liver. In addition, present knowledge of the role of GJ and HC in cell cycle progression, carcinogenesis and stem cell development is also discussed.

Gap junctions and hemichannels in signal transmission, function and development of bone ☆

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 1.2kDa including small ions, metabolites, ATP, prostaglandin and IP(3). 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. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

A novel role for MAP1 LC3 in nonautophagic cytoplasmic vacuolation death of cancer cells

Thiol reactive cyclopentenone prostaglandin, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), induced a novel, nonapoptotic and microtubule-associated protein 1 light chain 3 (MAP1 LC3) dependent but nonautophagic form of cell death in colon, breast and prostate cancer cell lines, characterized by extensive cytoplasmic vacuolation with dilatation of endoplasmic reticulum (ER). Disruption of sulfhydryl homeostasis, which resulted in ER stress, accumulation of ubiquitinated proteins and subsequent ER dilation, contributed to peroxisome proliferator-activated receptor γ (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 nonautophagic 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.

Glucocorticoid dose determines osteocyte cell fate

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.—Jia, J., Yao, W., Guan, M., Dai, W., Shahnazari, M., Kar, R., Bonewald, L., Jiang, J. X., Lane, N. E. Glucocorticoid dose determines osteocyte cell fate. FASEB J. 25, 3366–3376 (2011). www.fasebj.org

Direct Regulation of Osteocytic Connexin 43 Hemichannels through AKT Kinase Activated by Mechanical Stimulation

Background: Opening of Cx43 hemichannels by mechanical stress releases factors important for bone remodeling; however, the regulatory mechanism is unknown. Result: Upon mechanical stimulation, AKT phosphorylates integrin α5 and Cx43, increases interaction, and opens hemichannels. Conclusion: Phosphorylation of Cx43 and α5 by AKT is critical for hemichannel opening. Significance: This is the first report demonstrating the functional importance of AKT in regulation of Cx43 hemichannels. Connexin (Cx) 43 hemichannels in osteocytes are thought to play a critical role in releasing bone modulators in response to mechanical loading, a process important for bone formation and remodeling. However, the underlying mechanism that regulates the opening of mechanosensitive hemichannels is largely unknown. We have recently shown that Cx43 and integrin α5 interact directly with each other, and activation of PI3K appears to be required for Cx43 hemichannel opening by mechanical stimulation. Here, we show that mechanical loading through fluid flow shear stress (FFSS) increased the level of active AKT, a downstream effector of PI3K, which is correlated with the opening of hemichannels. Both Cx43 and integrin α5 are directly phosphorylated by AKT. Inhibition of AKT activation significantly reduced FFSS-induced opening of hemichannels and disrupted the interaction between Cx43 and integrin α5. Moreover, AKT phosphorylation on Cx43 and integrin α5 enhanced their interaction. In contrast to the C terminus of wild-type Cx43, overexpression of the C-terminal mutant containing S373A, a consensus site previously shown to be phosphorylated by AKT, failed to bind with α5 and hence could not inhibit hemichannel opening. Together, our results suggest that AKT activated by FFSS directly phosphorylates Cx43 and integrin α5, and Ser-373 of Cx43 plays a predominant role in mediating the interaction between these two proteins and Cx43 hemichannel opening, a crucial step to mediate the anabolic function of mechanical loading in the bone.

Connexin 43 Channels Protect Osteocytes Against Oxidative Stress–Induced Cell Death

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 show that H2O2 induced cell death of primary osteocytes and osteocytic MLO‐Y4 cells, and also caused dose‐dependent decreased 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‐old as opposed to the 5‐week‐old 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 that 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 small interfering RNA (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.

Osteocytic connexin hemichannels suppress breast cancer growth and bone metastasis

Although the skeleton is one of the 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.

Inhibition of mitochondrial division through covalent modification of Drp1 protein by 15 Deoxy- Δ12,14 - Prostaglandin J2

Arachidonic acid derived endogenous electrophile 15d-PGJ2 has gained much attention in recent years due to its potent anti-proliferative and anti-inflammatory actions mediated through thiol modification of cysteine residues in its target proteins. Here, we show that 15d-PGJ2 at 1 microM concentration converts normal mitochondria into large elongated and interconnected mitochondria through direct binding to mitochondrial fission protein Drp1 and partial inhibition of its GTPase activity. Mitochondrial elongation induced by 15d-PGJ2 is accompanied by increased assembly of Drp1 into large oligomeric complexes through plausible intermolecular interactions. The role of decreased GTPase activity of Drp1 in the formation of large oligomeric complexes is evident when Drp1 is incubated with a non-cleavable GTP analog, GTPgammaS or by a mutation that inactivated GTPase activity of Drp1 (K38A). The mutation of cysteine residue (Cys644) in the GTPase effector domain, a reported target for modification by reactive electrophiles, to alanine mimicked K38A mutation induced Drp1 oligomerization and mitochondrial elongation, suggesting the importance of cysteine in GED to regulate the GTPase activity and mitochondrial morphology. Interestingly, treatment of K38A and C644A mutants with 15d-PGJ2 resulted in super oligomerization of both mutant Drp1s indicating that 15d-PGJ2 may further stabilize Drp1 oligomers formed by loss of GTPase activity through covalent modification of middle domain cysteine residues. The present study documents for the first time the regulation of a mitochondrial fission activity by a prostaglandin, which will provide clues for understanding the pathological and physiological consequences of accumulation of reactive electrophiles during oxidative stress, inflammation and degeneration.

Oxidative stress induces phosphorylation of neuronal NOS in cardiomyocytes through AMP-activated protein kinase (AMPK)

Neuronal nitric oxide synthase (nNOS) plays a critical role in regulating cardiomyocyte function. nNOS was reported to decrease superoxide production in the myocardium by inhibiting the function of xanthine oxidoreductase. However, the effect of oxidative stress on nNOS in cardiomyocytes has not been determined. We report here that brief exposure of HL-1 cardiomyocytes to hydrogen peroxide (H2O2) induces phosphorylation of nNOS at serine 1412. This increase in phosphorylation was concomitant with increased nitric oxide (NO) production. Prolonged exposure to the oxidant, however, resulted in decreased expression of the protein. H2O2 treatment for short periods also stimulated phosphorylation of AKT and AMPK. H2O2-induced phosphorylation of nNOS was reduced when AMPK activity was inhibited by compound C, suggesting that AMPK is a mediator of oxidative stress-induced phosphorylation of nNOS. However, inhibition of AKT activity by the pan AKT inhibitor, AKTi, had no effect on nNOS phosphorylation caused by H2O2. These data demonstrate the novel regulation of nNOS phosphorylation and expression by oxidative stress.