Isra Darwech

IKKβ Activation Is Sufficient for RANK-Independent Osteoclast Differentiation and Osteolysis

Monocytes differentiate into osteoclasts through stimulation of receptor activator of NF‐κB (RANK). Many downstream effectors of RANK play a positive role in osteoclastogenesis, but their relative importance in osteoclast differentiation is unclear. We report the discovery that activation of a single pathway downstream of RANK is sufficient for osteoclast differentiation. In this regard, introduction of constitutively activated IKKβ (IKKβSSEE) but not wild‐type IKKβ into monocytes stimulates differentiation of bona fide osteoclasts in the absence of RANK ligand (RANKL). This phenomenon is independent of upstream signals because IKKβSSEE induced the development of bone‐resorbing osteoclasts from RANK and IKKα knockout monocytes and in conditions in which NEMO‐IKKβ association was inhibited. NF‐κB p100 and p105, but not RelB, were critical mediators of this effect. Inflammatory autocrine signaling by tumor necrosis factor α (TNF‐α) and interleukin 1 (IL‐1) were dispensable for the spontaneous osteoclastogenesis driven by IKKβSSEE. More important, adenoviral gene transfer of IKKβSSEE induced osteoclasts and osteolysis in calvariae and knees of mice. Our data establish the sufficiency of IKKβ activation for osteolysis and suggest that IKKβ hyperactivation may play a role in conditions of pathologic bone destruction refractory to RANK/RANKL proximal therapeutic interventions.

Role of the NF-κB axis in immune modulation of osteoclasts and bone loss

NF-κB is a vital component of the molecular programs for immune cell development and activation, inflammatory responses, and osteoclast differentiation. This transcriptional regulatory family is activated by diverse immunological and inflammatory stimuli and contributes to both positive feedback of the immune and osteolytic responses as well as their resolution. The ubiquilous expression of NF-κB components in osteoclasts and other immune cells creates an opportunity to gain a better understanding of the complex interplay between the immune and skeletal systems in physiological and pathological conditions and also makes NF-κB an important target in the treatment of autoimmune, inflammatory, and osteolytic diseases. Indeed, many genetic murine models have recently been developed which highlight the importance of NF-κB in basic processes including lymphocyte development, macrophage activation, and osteoclast differentiation. Furthermore, inhibition of NF-κB signaling has been demonstrated to ameliorate tissue inflammation and osteolysis in mouse models of inflammatory disease. A more complete understanding of the immunological factors that regulate NF-κB and the role that NF-κB plays in the immune and skeletal systems will elucidate potential avenues for intervening therapeutically in the pathological conditions of inflammation and osteolysis.

Tyrosine Phosphorylation Is Required for IκB Kinase-β (IKKβ) Activation and Function in Osteoclastogenesis

The transcription factor NF-κB is crucial for numerous cellular functions such as survival, differentiation, immunity, and inflammation. A key function of this family of transcription factors is regulation of osteoclast differentiation and function, which in turn controls skeletal homeostasis. The IκB kinase (IKK) complex, which contains IKKα, IKKβ, and IKKγ, is required for activation of NF-κB, and deletion of either IKKα or IKKβ resulted with defective osteoclast differentiation and survival. We have recently investigated the details of the mechanisms governing the role of IKKβ in osteoclastogenesis and found that constitutively active IKKβ in which serine residues 177/181 were mutated into negatively charged glutamic acids instigates spontaneous bona fide receptor activator of NF-κB ligand (RANKL)-independent osteoclastogenesis. To better understand and define the functional role of IKKβ domains capable of regulating the osteoclastogenic activity of IKK, we investigated key motifs in the activation T loop of IKKβ, which are potentially capable of modulating its osteoclastogenic activity. We discovered that dual serine (traditional serine residues 177/181) and tyrosine (188/199) phosphorylation events are crucial for IKKβ activation. Mutation of the latter tyrosine residues blunted the NF-κB activity of wild type and constitutively active IKKβ, and tyrosine 188/199-deficient IKKβ inhibited osteoclastogenesis. Thus, tyrosines 188/199 are a novel target for regulating IKKβ activity, at least in osteoclasts.

Impediment of NEMO oligomerization inhibits osteoclastogenesis and osteolysis.

The transcription factor NF‐κB is essential for osteoclastogenesis and is considered an immune‐modulator of rheumatoid arthritis and inflammatory osteolysis. Activation of NF‐κB subunits is regulated by the upstream IκB kinase (IKK) complex which contains IKKα, IKKβ, and IKKγ; the latter also known as NF‐κB essential modulator (NEMO). The role of IKKα and IKKβ in the skeletal development and inflammatory osteolysis has been described, whereas little is known regarding the role of NEMO in this setting. Typically, signals induced by RANK ligand (RANKL) or TNF prompt oligomerization of NEMO monomers through the coiled‐coil‐2 (CC2) and leucine zipper (LZ) motifs. This step facilitates binding to IKKs and further relaying signal transduction. Given the central role of NF‐κB in osteoclastogenesis, we asked whether NEMO is essential for osteoclastogenesis and whether interruption of NEMO oligomerization impedes osteoclast differentiation in vitro and in vivo. Using cell‐permeable short peptides overlapping the CC2 and LZ motifs we show that these peptides specifically bind to NEMO monomers, prevent trimer formation, and render NEMO monomers susceptible for ubiquitin‐mediated degradation. Further, CC2 and LZ peptides attenuate RANKL‐ and TNF‐induced NF‐κB signaling in bone marrow‐derived osteoclast precursors (OCPs). More importantly, these peptides potently inhibit osteoclastogenesis, in vitro, and arrest RANKL‐induced osteolysis, in mice. To further ascertain its role in osteoclastogenesis, we were able to block osteoclastogenesis using NEMO siRNA knockdown approach. Collectively, our data establish that obstruction of NEMO oligomerization destabilizes NEMO monomers, inhibits NF‐κB activation, impedes osteoclastogenesis and arrests inflammatory osteolysis. Thus, NEMO presents itself as a promising target for anti‐osteolytic intervention. J. Cell. Biochem. 108: 1337–1345, 2009.

Stromal cells participate in the murine esophageal mucosal injury response.

We identified α-smooth muscle actin (α-SMA)- and vimentin-expressing spindle-shaped esophageal mesenchymal cells in the adult and neonate murine esophageal lamina propria. We hypothesized that these esophageal mesenchymal cells express and secrete signaling and inflammatory mediators in response to injury. We established primary cultures of esophageal mesenchymal cells using mechanical and enzymatic digestion. We demonstrate that these primary cultures are nonhematopoietic, nonendothelial, stromal cells with myofibroblast-like features. These cells increase secretion of IL-6 in response to treatment with acidified media and IL-1β. They also increase bone morphogenetic protein (Bmp)-4 secretion in response to sonic hedgehog. The location of these cells and their biological functions demonstrate their potential role in regulating esophageal epithelial responses to injury and repair.

Epimorphin(-/-) mice are protected, in part, from acute colitis via decreased interleukin 6 signaling.

Epimorphin (Epim), a member of the syntaxin family of membrane-bound, intracellular vesicle-docking proteins, is expressed in intestinal myofibroblasts and macrophages. We demonstrated previously that Epimorphin(-/-)(Epim(-/-)) mice are protected, in part, from dextran sodium sulfate (DSS)-induced colitis. Although interleukin (IL)-6/p-Stat3 signaling has been implicated in the pathogenesis of colitis, the myofibroblast contribution to IL-6 signaling in colitis remains unexplored. Our aim was to investigate the IL-6 pathway in Epim(-/-) mice in the DSS colitis model. Whole colonic tissue, epithelium, and stroma of WT and congenic Epim(-/-) mice treated with 5% DSS for 7 days were analyzed for IL-6 and a downstream effector, p-Stat3, by immunostaining and immunoblot. Colonic myofibroblast and peritoneal macrophage IL-6 secretion were evaluated by enzyme-linked immunosorbent assay. IL-6 and p-Stat3 expression were decreased in Epim(-/-) vs WT colon. A relative increase in stromal vs epithelial p-Stat3 expression was observed in WT mice but not in Epim(-/-) mice. Epim deletion abrogates IL-6 secretion from colonic myofibroblasts treated with IL-1β and decreases IL-6 secretion from peritoneal macrophages in a subset of DSS-treated mice. Epim deletion inhibits IL-6 secretion most profoundly from colonic myofibroblasts. Distribution of Stat3 activation is altered in DSS-treated Epim(-/-) mice. Our findings support the notion that myofibroblasts modulate IL-6/p-Stat3 signaling in DSS-treated Epim(-/-) mice.

Vitamin D3 supplementation decreases a unique circulating monocyte cholesterol pool in patients with type 2 diabetes

Cross-sectional studies indicate consistent associations between low 25(OH)D concentration and increased risk of cardiovascular disease (CVD), but results of randomized control trials (RCTs) are mixed. However, the majority of the RCTs do not focus on type 2 diabetics, potentially obscuring the effects of vitamin D in this population. In vitro 1,25(OH)2D3 downregulates macrophage cholesterol deposition, but the in vivo effects are unknown. To explore potential mechanisms of the effects of vitamin D on CVD risk in patients with type 2 diabetes, we isolated monocytes in a subset of 26 patients from our RCT of diabetics with baseline serum 25(OH)D <25ng/mL randomized to vitamin D3 4000 IU/day or placebo for 4 months. Upon enrollment, the mean 25(OH)D level was 17ng/mL, which increased to 36ng/mL after vitamin D and remained unchanged in the placebo group. Before randomization, groups demonstrated similar mean hemoglobin A1c and plasma lipids levels, none of which was significantly altered by vitamin D supplementation. Moreover, assessment of oxidized LDL uptake in monocytes cultured in the patients own serum before vs. after treatment resulted in >50% reduction in the vitamin D group with no change in the placebo group. This was mediated through suppression of endoplasmic reticulum stress and scavenger receptor CD36 protein expression. The reduction in monocyte cholesterol uptake was reflected in a 19% decrease in total monocyte cholesterol content. Interestingly, cross-sectional analysis of circulating monocytes from vitamin D-deficient vs. sufficient diabetic patients revealed 8-fold higher cholesteryl ester content, confirming the capacity of these monocytes to uptake and carry cholesterol in the circulation. This study identifies a unique circulating cholesterol pool within monocytes that is modulated by vitamin D and has the potential to contribute to CVD in type 2 diabetes.

Deletion of JNK2 prevents vitamin-D-deficiency-induced hypertension and atherosclerosis in mice

The c-Jun N-terminal kinase 2 (JNK2) signaling pathway contributes to inflammation and plays a key role in the development of obesity-induced insulin resistance and cardiovascular disease. Macrophages are key cells implicated in these metabolic abnormalities. Active vitamin D downregulates macrophage JNK activation, suppressing oxidized LDL cholesterol uptake and foam cell formation and promoting an anti-inflammatory phenotype. To determine whether deletion of JNK2 prevents high blood pressure and atherosclerosis known to be induced by vitamin D deficiency in mice, we generated mice with knockout of JNK2 in a background susceptible to diet-induced atherosclerosis (LDLR-/-). JNK2-/- LDLR-/- and LDLR-/- control mice were fed vitamin D-deficient chow for 8 weeks followed by vitamin D-deficient high fat diet (HFD) for 10 weeks and assessed before and after HFD. There was no difference in fasting glucose, cholesterol, triglycerides, or free fatty acid levels. However, JNK2-/- mice, despite vitamin D-deficient diet, had 20-30mmHg lower systolic (SBP) and diastolic (DBP) blood pressure before HFD compared to control mice fed vitamin D-deficient diets, with persistent SBP differences after HFD. Moreover, deletion of JNK2 reduced HFD-induced atherosclerosis by 30% in the proximal aorta when compared to control mice fed vitamin D-deficient diets. We have previously shown that peritoneal macrophages obtained from LDLR-/- mice fed vitamin D-deficient HFD diets have higher foam cell formation compared to those from mice on vitamin D-sufficient HFD. The increased total cellular cholesterol and modified cholesterol uptake in macrophages from mice on vitamin D-deficient HFD were blunted by deletion of JNK2. These data suggest that JNK2 signaling activation is necessary for the atherosclerosis and hypertension induced by vitamin D deficiency.