Circadian Rhythm Perturbation Aggravates Gut Microbiota Dysbiosis in Dextran Sulfate Sodium-Induced Colitis in Mice.

Circadian rhythm disruption is increasingly considered an environmental risk factor for the development and exacerbation of inflammatory bowel disease. We have reported in a previous study that nychthemeral dysregulation is associated with an increase in intestinal barrier permeability and inflammation in mice with dextran sulfate sodium (DSS)-induced colitis. To investigate the effect of circadian rhythm disruption on the composition and diversity of the gut microbiota (GM), sixty male C57BL/6J mice were initially divided to two groups, with the shifted group (n = 30) exposed to circadian shifts for three months and the non-shifted group (n = 30) kept under a normal light-dark cycle. The mice of the shifted group were cyclically housed for five days under the normal 12:12 h light-dark cycle, followed by another five days under a reversed light-dark cycle. At the end of the three months, a colitis was induced by 2% DSS given in the drinking water of 30 mice. Animals were then divided into four groups (n = 15 per group): sham group non-shifted (Sham-NS), sham group shifted (Sham-S), DSS non-shifted (DSS-NS) and DSS shifted (DSS-S). Fecal samples were collected from rectal content to investigate changes in GM composition via DNA extraction, followed by high-throughput sequencing of the bacterial 16S rRNA gene. The mouse GM was dominated by three phyla: Firmicutes, Bacteroidetes and Actinobacteria. The Firmicutes/Bacteroidetes ratio decreased in mice with induced colitis. The richness and diversity of the GM were reduced in the colitis group, especially in the group with inverted circadian rhythm. Moreover, the GM composition was modified in the inverted circadian rhythm group, with an increase in Alloprevotella, Turicibacter, Bacteroides and Streptococcus genera. Circadian rhythm inversion exacerbates GM dysbiosis to a less rich and diversified extent in a DSS-induced colitis model. These findings show possible interplay between circadian rhythm disruption, GM dynamics and colitis pathogenesis.

Artificial Intelligence, Computational Simulations, and Extended Reality in Cardiovascular Interventions.

Artificial intelligence, computational simulations, and extended reality, among other 21st century computational technologies, are changing the health care system. To collectively highlight the most recent advances and benefits of artificial intelligence, computational simulations, and extended reality in cardiovascular therapies, we coined the abbreviation AISER. The review particularly focuses on the following applications of AISER: 1) preprocedural planning and clinical decision making; 2) virtual clinical trials, and cardiovascular device research, development, and regulatory approval; and 3) education and training of interventional health care professionals and medical technology innovators. We also discuss the obstacles and constraints associated with the application of AISER technologies, as well as the proposed solutions. Interventional health care professionals, computer scientists, biomedical engineers, experts in bioinformatics and visualization, the device industry, ethics committees, and regulatory agencies are expected to streamline the use of AISER technologies in cardiovascular interventions and medicine in general.

Increasing surgeon experience and cumulative institutional experience drive decreasing hospital mortality after reoperative cardiac surgery.

OBJECTIVE: The study objective was to identify the effects of surgeon experience and age, in the context of cumulative institutional experience, on risk-adjusted hospital mortality after cardiac reoperations. METHODS: From 1951 to 2020, 36 surgeons performed 160,338 cardiac operations, including 32,871 reoperations. Hospital death was modeled using a novel tree-bagged, generalized varying-coefficient method with 6 variables reflecting cumulative surgeon and institutional experience up to each cardiac operation: (1) number of total and (2) reoperative cardiac operations performed by a surgeon, (3) cumulative institutional number of total and (4) reoperative cardiac operations, (5) year of surgery, and (6) surgeon age at each operation. These were adjusted for 46 patient characteristics and surgical components. RESULTS: There were 1470 hospital deaths after cardiac reoperations (4.5%). At the institutional level, hospital death decreased exponentially and became less variable, leveling at 1.2% after approximately 14,000 cardiac reoperations. For all surgeons as a group, hospital death decreased rapidly over the first 750 reoperations and then gradually decreased with increasing experience to less than 1% after approximately 4000 reoperations. Surgeon age up to 75 years was associated with ever-decreasing hospital death. CONCLUSIONS: Surgeon age and experience have been implicated in adverse surgical outcomes, particularly after complex cardiac operations, with young surgeons being novices and older surgeons having declining ability. However, at Cleveland Clinic, outcomes of cardiac reoperations improved with increasing primary surgeon experience, without any suggestion to mid-70s of an age cutoff. Patients were protected by the cumulative background of institutional experience that created a culture of safety and teamwork that mitigated adverse events after cardiac surgery.

TRPC3 Regulates Islet Beta-Cell Insulin Secretion.

Insulin release is tightly controlled by glucose-stimulated calcium (GSCa) through hitherto equivocal pathways. This study investigates TRPC3, a non-selective cation channel, as a critical regulator of insulin secretion and glucose control. TRPC3's involvement in glucose-stimulated insulin secretion (GSIS) is studied in human and animal islets. TRPC3-dependent in vivo insulin secretion is investigated using pharmacological tools and Trpc3-/- mice. TRPC3's involvement in islet glucose uptake and GSCa is explored using fluorescent glucose analogue 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose and calcium imaging. TRPC3 modulation by a small-molecule activator, GSK1702934A, is evaluated in type 2 diabetic mice. TRPC3 is functionally expressed in human and mouse islet beta cells. TRPC3-controlled insulin secretion is KATP -independent and primarily mediated by diacylglycerol channel regulation of the cytosolic calcium oscillations following glucose stimulation. Conversely, glucose uptake in islets is independent of TRPC3. TRPC3 pharmacologic inhibition and knockout in mice lead to defective insulin secretion and glucose intolerance. Subsequently, TRPC3 activation through targeted small-molecule enhances insulin secretion and alleviates diabetes hallmarks in animals. This study imputes a function for TRPC3 at the onset of GSIS. These insights strengthen one's knowledge of insulin secretion physiology and set forth the TRPC3 channel as an appealing candidate for drug development in the treatment of diabetes.

Triggering receptor expressed on myeloid cells-1 (TREM-1) inhibition in atherosclerosis.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a transmembrane protein expressed on endothelial cells, white blood cells, smooth muscle cells and platelets. TREM-1 plays an important role in innate immunity. TREM-1 activation pathways are implicated both in sepsis and in non-infectious inflammatory conditions, including atherosclerosis. TREM-1 enhances the subendothelial lipid accumulation and expression of pro-inflammatory cytokines and matrix-degrading enzymes, thereby promoting inflammation and plaque destabilization. TREM-1 inhibitors attenuate the inflammatory process in the atherosclerotic plaque, leading to plaque stabilization. This review focuses on the role of TREM-1 in the pathophysiology of atherosclerosis and the effects of TREM-1 inhibition in the natural history of the disease.

Blood Donation in Lebanon: A Six-Year Retrospective Study of a Decentralized Fragmented Blood Management System.

Introduction In developing countries, the lack of a sufficient and safe blood supply is a significant impediment to providing health care. Lebanon is notable for its absence of a Donor Management System to ensure continuous donor recruitment and scheduling. Herein, we report the findings of Lebanon's first large retrospective population-based study to investigate blood types and donation that is critical for managing community blood supply. Methods The non-remunerated voluntary blood donors were recruited by the non-profit organization "Donner Sang Compter". The study spanned six years, from August 2015 to May 2021, and included 36,002 people from 18 districts throughout Lebanon's nine governorates. Results The most prevalent blood type was A (42%), followed by O (37.48%), B (13.86%), and the AB group (6.84%). RhD+ groups were predominant (88.45%), with A+ being the most (37.84%) and AB- being the least prevalent (1.05%). Furthermore, blood type and donation profiling revealed a substantial geographical variation in the frequency of blood groups, despite the relatively small country's area. As for blood donation, when gender and age were considered, young male donors dominated the pool across the country. Conclusion This study on blood type prevalence and blood donor demographics may pave the way for the development of a more coherent and integrated blood management system in Lebanon, as opposed to the fragmented and decentralized system now in existence. These findings also provide crucial clinical information for the country's future transfusion medicine policies and practices, which is vital in such a precarious part of the world.

Comparative study between prison- and community-based treatment satisfaction for opioid use disorder in Lebanon.

PURPOSE: Opioid substitution treatment (OST), such as Buprenorphine, has become a well-established evidence-based approach for the treatment of inmates with opioid use disorder (OUD) in most of the developed world. However, its application in Lebanon remains mainly as a community-based intervention. The purpose of this paper is to highlight the need of its implementation within the Lebanese correctional system. DESIGN/METHODOLOGY/APPROACH: The work is a pilot cross-sectional study that compares two groups: 30 male adult prisoners with OUD convictions receiving symptomatic treatment and 30 male adult community patients with OUD receiving Buprenorphine. The objective was to measure the difference in the patients' general perception and satisfaction of the treatments available. OUD was diagnosed using the Diagnostic and Statistical Manual of Mental Disorders 5th Edition criteria and the level of satisfaction was measured by "Treatment Perceptions Questionnaire (TPQ)." FINDINGS: The prison group reported significantly lower satisfaction when compared to the community group (total TPQ mean scores: M=34.73, SD =4.12 and M=16.67, SD =4.78, respectively, with t (56.76) =15.68, p=0.000). Furthermore, age, marital status, education level and elapsed time in treatment had no significant interactions with the total TPQ score. ORIGINALITY/VALUE: The major principles of the ethics of care and evidence-based safe practices will be proposed for the introduction of Buprenorphine to Lebanese prisons. This work provides an opportunity for the expansion of the Lebanese OST program and consequently other countries in the region could benefit from this experience.

Specific RANK Cytoplasmic Motifs Drive Osteoclastogenesis.

Upon receptor activator of NF-κB ligand (RANKL) binding, RANK promotes osteoclast formation through the recruitment of tumor necrosis factor (TNF) receptor-associated factors (TRAFs). In vitro assays identified two RANK intracellular motifs that bind TRAFs: PVQEET560-565 (Motif 2) and PVQEQG604-609 (Motif 3), which potently mediate osteoclast formation in vitro. To validate the in vitro findings, we have generated knock-in (KI) mice harboring inactivating mutations in RANK Motifs 2 and 3. Homozygous KI (RANKKI/KI ) mice are born at the predicted Mendelian frequency and normal in tooth eruption. However, RANKKI/KI mice exhibit significantly more trabecular bone mass than age- and sex-matched heterozygous KI (RANK+/KI ) and wild-type (RANK+/+ ) counterparts. Bone marrow macrophages (BMMs) from RANKKI/KI mice do not form osteoclasts when they are stimulated with macrophage colony-stimulating factor (M-CSF) and RANKL in vitro. RANKL is able to activate the NF-κB, ERK, p38, and JNK pathways in RANKKI/KI BMMs, but it cannot stimulate c-Fos or NFATc1 in the RANKKI/KI cells. Previously, we showed that RANK signaling plays an important role in Porphyromonas gingivalis (Pg)-mediated osteoclast formation by committing BMMs into the osteoclast lineage. Here, we show that RANKL-primed RANKKI/KI BMMs are unable to differentiate into osteoclasts in response to Pg stimulation, indicating that the two RANK motifs are required for Pg-induced osteoclastogenesis. Mechanistically, RANK Motifs 2 and 3 facilitate Pg-induced osteoclastogenesis by stimulating c-Fos and NFATc1 expression during the RANKL pretreatment phase as well as rendering c-Fos and NFATc1 genes responsive to subsequent Pg stimulation. Cell-penetrating peptides (CPPs) conjugated with RANK segments containing Motif 2 or 3 block RANKL- and Pg-mediated osteoclastogenesis. The CPP conjugates abrogate RANKL-stimulated c-Fos and NFATc1 expression but do not affect RANKL-induced activation of NF-κB, ERK, p38, JNK, or Akt signaling pathway. Taken together, our current findings demonstrate that RANK Motifs 2 and 3 play pivotal roles in osteoclast formation in vivo and mediate Pg-induced osteoclastogenesis in vitro.

Circadian Rhythm Disruption Aggravates DSS-Induced Colitis in Mice with Fecal Calprotectin as a Marker of Colitis Severity.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic immunologically mediated pathology that remains a major health burden. Circadian rhythm disruption leads to a deregulation in the immune system which is a major risk factor for IBD. AIMS: Since fecal calprotectin (FC) has been a useful tool for monitoring IBD, we aimed to evaluate the effect of circadian rhythm alteration on gut inflammation status and whether FC is associated with the severity of colitis. METHODS: C57BL/6J mice were exposed to circadian shifts for 3 months, and then colitis was induced by 2% dextran sulfate sodium (DSS). Colitis was evaluated according to clinical symptoms and histological scoring. Plasma and intestinal inflammatory and permeability markers as well as fecal and intestinal calprotectin were assessed. RESULTS: Circadian shifts aggravated DSS-induced colitis with increased diarrhea, flatulence, and fecal blood associated with decreased colon length. In addition, intestinal cryptic architecture was lost with the presence of increased inflammation, mucosal muscle thickening, and cryptic abscesses. Plasma tumor necrosis factor alpha, interleukin 1 beta, interleukin 6, and C-reactive protein upregulations were paralleled by the deterioration of intestinal permeability. Calprotectin expression and distribution increased in the intestines and feces of shifted animals, and levels highly correlated with the increases in intestinal inflammation and permeability. CONCLUSIONS: Circadian rhythm disruption aggravates DSS-induced colitis, whereas fecal and intestinal calprotectin associates with the severity of disease. Calprotectin might be a useful marker and tool for assessing patients at risk of IBD due to lifestyles with disruptive sleep patterns.

VEGF-C attenuates renal damage in salt-sensitive hypertension.

Salt-sensitive hypertension is a major risk factor for renal impairment leading to chronic kidney disease. High-salt diet leads to hypertonic skin interstitial volume retention enhancing the activation of the tonicity-responsive enhancer-binding protein (TonEBP) within macrophages leading to vascular endothelial growth factor C (VEGF-C) secretion and NOS3 modulation. This promotes skin lymphangiogenesis and blood pressure regulation. Whether VEGF-C administration enhances renal and skin lymphangiogenesis and attenuates renal damage in salt-sensitive hypertension remains to be elucidated. Hypertension was induced in BALB/c mice by a high-salt diet. VEGF-C was administered subcutaneously to high-salt-treated mice as well as control animals. Analyses of kidney injury, inflammation, fibrosis, and biochemical markers were performed in vivo. VEGF-C reduced plasma inflammatory markers in salt-treated mice. In addition, VEGF-C exhibited a renal anti-inflammatory effect with the induction of macrophage M2 phenotype, followed by reductions in interstitial fibrosis. Antioxidant enzymes within the kidney as well as urinary RNA/DNA damage markers were all revelatory of abolished oxidative stress under VEGF-C. Furthermore, VEGF-C decreased the urinary albumin/creatinine ratio and blood pressure as well as glomerular and tubular damages. These improvements were associated with enhanced TonEBP, NOS3, and lymphangiogenesis within the kidney and skin. Our data show that VEGF-C administration plays a major role in preserving renal histology and reducing blood pressure. VEGF-C might constitute an interesting potential therapeutic target for improving renal remodeling in salt-sensitive hypertension.

CoreValve Double Jeopardy: Embolized Valve Capture With Subsequent Valve.

During transcatheter aortic valve replacement with a self-expanding prosthesis, prosthesis embolization represents a rare but severe complication. Etiologies of prosthesis embolization include improper sizing and malpositioning, specifically high deployment with respect to the aortic annulus. Treatment of embolization into the aorta relies upon repositioning of the prosthesis using endovascular snares or removal with open surgery. Patients with prosthesis embolization have a high risk of mortality and morbidity including stroke and aortic dissection associated with manipulation of the prosthesis in the ascending aorta. We describe a case of self-expanding prosthesis embolization and present a solution using a second prosthesis to capture the embolized one.

Mifepristone reduces hypothalamo-pituitary-adrenal axis activation and restores weight loss in rats subjected to dietary restriction and methylphenidate administration.

This study evaluates the efficacy of mifepristone on weight restoration in rats subjected to dietary restriction and methylphenidate administration. 25 female rats aged between 9 and 12 months were divided into 2 groups: 5 controls (exposed only to dietary restriction) and 20 rats that were administered 5 mg/kg/d of methylphenidate before meal exposure, for 36 days. Among rats who responded to methylphenidate (weight loss of 15-25%) weeks after its administration, a group of 6 rats continued to receive only methylphenidate ("Met" group), and another group received 10 mg/kg/d of mifepristone in addition to methylphenidate for 18 days ("Met+Mif" group; n = 6). The mean weight of the "Met+Mif" group remained significantly lower when compared to the control group (87.63 ±â€¯2.83% vs 96.29 ±â€¯3.26%; p < 0.001 respectively) but was significantly higher than that of the "Met" group (87.63 ±â€¯2.83% vs. 80.61 ±â€¯3.52%; p < 0.001 respectively). Plasma concentrations of adiponectin and gene expression of its receptors in rats brain were significantly higher in the "Met" group as compared to the "Met+Mif" and control groups (p < 0.01). Accordingly, mifepristone reduces HPA axis activation and restores weight through adipose tissue recovering. It might be considered a promising treatment for anorexia nervosa patients in future studies.

C/EBPα transcription factor is regulated by the RANK cytoplasmic 535IVVY538 motif and stimulates osteoclastogenesis more strongly than c-Fos.

Binding of receptor activator of NF-κB ligand (RANKL) to its receptor RANK on osteoclast (OC) precursors up-regulates c-Fos and CCAAT/enhancer-binding protein-α (C/EBPα), two critical OC transcription factors. However, the effects of c-Fos and C/EBPα on osteoclastogenesis have not been compared. Herein, we demonstrate that overexpression of c-Fos or C/EBPα in OC precursors up-regulates OC genes and initiates osteoclastogenesis independently of RANKL. However, although C/EBPα up-regulated c-Fos, c-Fos failed to up-regulate C/EBPα in OC precursors. Consistently, C/EBPα overexpression more strongly promoted OC differentiation than did c-Fos overexpression. RANK has a cytoplasmic 535IVVY538 (IVVY) motif that is essential for osteoclastogenesis, and we found that mutation of the IVVY motif blocked OC differentiation by partly inhibiting expression of C/EBPα but not expression of c-Fos. We therefore hypothesized that C/EBPα overexpression might rescue osteoclastogenesis in cells expressing the mutated IVVY motif. However, overexpression of C/EBPα or c-Fos failed to stimulate osteoclastogenesis in the mutant cells. Notably, the IVVY motif mutation abrogated OC gene expression compared with a vector control, suggesting that the IVVY motif might counteract OC inhibitors during osteoclastogenesis. Consistently, the IVVY motif mutant triggered up-regulation of recombinant recognition sequence-binding protein at the Jκ site (RBP-J) protein, a potent OC inhibitor. Mechanistically, C/EBPα or c-Fos overexpression in the mutant cells failed to control the up-regulated RBP-J expression, leading to suppression of OC genes. Accordingly, RBP-J silencing in the mutant cells rescued osteoclastogenesis with C/EBPα or c-Fos overexpression with C/EBPα exhibiting a stronger osteoclastogenic effect. Collectively, our findings indicate that C/EBPα is a stronger inducer of OC differentiation than c-Fos, partly via C/EBPα regulation by the RANK 535IVVY538 motif.

Monocyte-Specific Knockout of C/ebpα Results in Osteopetrosis Phenotype, Blocks Bone Loss in Ovariectomized Mice, and Reveals an Important Function of C/ebpα in Osteoclast Differentiation and Function.

CCAAT/enhancer-binding protein α (C/ebpα) is critical for osteoclastogenesis by regulating osteoclast (OC) lineage commitment and is also important for OC differentiation and function in vitro. However, the role of C/ebpα in postnatal skeletal development has not been reported owing to lethality in C/ebpα-/- mice from hypoglycemia within 8 hours after birth. Herein, we generated conditional knockout mice by deleting the C/ebpα gene in monocyte via LysM-Cre to examine its role in OC differentiation and function. C/ebpαf/f LysM-Cre mice exhibited postnatal osteopetrosis due to impaired osteoclastogenesis, OC lineage priming defects, as well as defective OC differentiation and activity. Furthermore, our ex vivo analysis demonstrated that C/ebpα conditional deletion significantly reduced OC differentiation, maturation, and activity while mildly repressing macrophage development. At the molecular level, C/ebpα deficiency significantly suppresses the expressions of OC genes associated with early stages of osteoclastogenesis as well as genes associated with OC differentiation and activity. We also identified numerous C/ebpα critical cis-regulatory elements on the Cathepsin K promoter that allow C/ebpα to significantly upregulate Cathepsin K expression during OC differentiation and activity. In pathologically induced mouse model of osteoporosis, C/ebpα deficiency can protect mice against ovariectomy-induced bone loss, uncovering a central role for C/ebpα in osteolytic diseases. Collectively, our findings have further established C/ebpα as a promising therapeutic target for bone loss by concurrently targeting OC lineage priming, differentiation, and activity. © 2017 American Society for Bone and Mineral Research.

C/EBPα and PU.1 exhibit different responses to RANK signaling for osteoclastogenesis.

The transcription factors C/EBPα and PU.1 are upregulated by RANKL through activation of its receptor RANK during osteoclastogenesis and are critical for osteoclast differentiation. Herein we investigated the mechanisms underlying how C/EBPα and PU.1 regulate osteoclast differentiation in response to RANK signaling. We showed that C/EBPα or PU.1 overexpression could initiate osteoclastogenesis and upregulate the expressions of the osteoclast genes encoding the nuclear factor of activated T-cells, C1, cathepsin K, and tartrate-resistant acid phosphatase independently of RANKL. However, while PU.1 upregulated C/EBPα, C/EBPα could not upregulate PU.1. RANK has a unique cytoplasmic domain, 535IVVY538 motif, which is crucial for osteoclast differentiation. We demonstrated that mutational inactivation of RANK IVVY motif blocked osteoclast differentiation and significantly attenuated C/EBPα, but not PU.1, expression, indicating that RANK-IVVY-induced signaling is dispensable to PU.1 upregulation during osteoclastogenesis. However, C/EBPα or PU.1 overexpression failed to promote osteoclastogenesis in cells expressing mutated RANK IVVY motif. We noted that RANK-IVVY-motif inactivation significantly repressed osteoclast genes as compared with a vector control, suggesting that IVVY motif might also negatively regulate osteoclast inhibitors during osteoclastogenesis. Consistently, IVVY-motif inactivation triggered upregulation of RBP-J, a potent osteoclast inhibitor, during osteoclastogenesis. Notably, C/EBPα or PU.1 overexpression in cells expressing mutated RANK IVVY motif failed to control the deregulated RBP-J expression, resulting in repression of osteoclast genes. Accordingly, RBP-J silencing in the mutant cells rescued osteoclastogenesis with C/EBPα or PU.1 overexpression. In conclusion, we revealed that while PU.1 and C/EBPα are critical for osteoclastogenesis, they respond differently to RANKL-induced activation of RANK IVVY motif.

An optimized protocol for purification of functional islets of Langerhans.

Islets of Langerhans and ß-cell isolation constitute routinely used cell models for diabetic research, and refining islet isolation protocols and cell quality assessment is a high priority. Numerous protocols have been published describing isolate of islets, but often rigorous and systematic assessment of their integrity is lacking. Herein, we propose a new protocol for optimal generation of islets. Pancreases from mice and rats were excised and digested using a low-activity collagenase solution and islets were then purified by a series of sedimentations and a Percoll gradient. Islets were maintained in culture for 5 days, during which viability, pro/antiapoptotic, and islet-specific genes, glucose-stimulated calcium entry, glucose uptake, and insulin secretion were assessed. The commonly used islet isolation technique by collagenase injection through the common bile duct (CBD) was also performed and compared with the present approach. This new protocol produced islets that retained a healthy status as demonstrated by the yield of stable living cells. Furthermore, calcium oscillation, glucose uptake, and insulin secretion remained intact in the islet cultures. This was reproducible when many rodent species were used, and neither sex nor age affected the cells behavior. When compared with the CBD technique, islet physiology was similar. Finally, this approach was used to uncover new ion channel candidates implicated in insulin secretion. In conclusion, this study outlines an efficient protocol for islet preparation that may support research into new therapeutic targets in diabetes research.

CCAAT/Enhancer-binding Protein α (C/EBPα) Is Important for Osteoclast Differentiation and Activity.

CCAAT/enhancer-binding protein (C/EBPα) can appoint mouse bone marrow (MBM) cells to the osteoclast (OC) lineage for osteoclastogenesis. However, whether C/EBPα is also involved in OC differentiation and activity is unknown. Here we demonstrated that C/EBPα overexpression in MBM cells can promote OC differentiation and strongly induce the expression of the OC genes encoding the nuclear factor of activated T-cells, c1 (NFATc1), cathepsin K (Cstk), and tartrate-resistant acid phosphatase 5 (TRAP) with receptor activator of NF-κB ligand-evoked OC lineage priming. Furthermore, while investigating the specific stage of OC differentiation that is regulated by C/EBPα, our gene overexpression studies revealed that, although C/EBPα plays a stronger role in the early stage of OC differentiation, it is also involved in the later stage. Accordingly, C/EBPα knockdown drastically inhibits osteoclastogenesis and markedly abrogates the expression of NFATc1, Cstk, and TRAP during OC differentiation. Consistently, C/EBPα silencing revealed that, although lack of C/EBPα affects all stages of OC differentiation, it has more impact on the early stage. Importantly, we showed that ectopic expression of rat C/EBPα restores osteoclastogenesis in C/EBPα-depleted MBM cells. Furthermore, our subsequent functional assays showed that C/EBPα exhibits a dispensable role on actin ring formation by mature OCs but is critically involved in bone resorption by stimulating extracellular acidification and regulating cell survival. We revealed that C/EBPα is important for receptor activator of NF-κB ligand-induced Akt activation, which is crucial for OC survival. Collectively, these results indicate that C/EBPα functions throughout osteoclastogenesis as well as in OC function. This study provides additional understanding of the roles of C/EBPα in OC biology.

Regulation of Receptor for Advanced Glycation End Products (RAGE) Ectodomain Shedding and Its Role in Cell Function.

The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor that can undergo proteolysis at the cell surface to release a soluble ectodomain. Here we observed that ectodomain shedding of RAGE is critical for its role in regulating signaling and cellular function. Ectodomain shedding of both human and mouse RAGE was dependent on ADAM10 activity and induced with chemical activators of shedding (ionomycin, phorbol 12-myristate 13-acetate, and 4-aminophenylmercuric acetate) and endogenous stimuli (serum and RAGE ligands). Ectopic expression of the splice variant of RAGE (RAGE splice variant 4), which is resistant to ectodomain shedding, inhibited RAGE ligand dependent cell signaling, actin cytoskeleton reorganization, cell spreading, and cell migration. We found that blockade of RAGE ligand signaling with soluble RAGE or inhibitors of MAPK or PI3K blocked RAGE-dependent cell migration but did not affect RAGE splice variant 4 cell migration. We finally demonstrated that RAGE function is dependent on secretase activity as ADAM10 and γ-secretase inhibitors blocked RAGE ligand-mediated cell migration. Together, our data suggest that proteolysis of RAGE is critical to mediate signaling and cell function and may therefore emerge as a novel therapeutic target for RAGE-dependent disease states.

The IVVY Motif and Tumor Necrosis Factor Receptor-associated Factor (TRAF) Sites in the Cytoplasmic Domain of the Receptor Activator of Nuclear Factor κB (RANK) Cooperate to Induce Osteoclastogenesis.

Receptor activator of NF-κB (RANK) activation by RANK ligand (RANKL) mediates osteoclastogenesis by recruiting TNF receptor-associated factors (TRAFs) via three cytoplasmic motifs (motif 1, PFQEP(369-373); motif 2, PVQEET(559-564); and motif 3, PVQEQG(604-609)) to activate the NF-κB and MAPK signaling pathways. RANK also has a TRAF-independent motif (IVVY(535-538)), which is dispensable for the activation of TRAF-induced signaling pathways but essential for osteoclast lineage commitment by inducing the expression of nuclear factor of activated T-cells c1 (NFATc1) to regulate osteoclast gene expression. Notably, TNF/IL-1-mediated osteoclastogenesis requires RANK ligand assistance, and the IVVY motif is also critical for TNF/IL-1-mediated osteoclastogenesis by rendering osteoclast genes responsive to these two cytokines. Here we show that the two types of RANK cytoplasmic motifs have to be on the same RANK molecule to mediate osteoclastogenesis, suggesting a functional cooperation between them. Subsequent osteoclastogenesis assays with TNF or IL-1 revealed that, although all three TRAF motifs play roles in TNF/IL-1-mediated osteoclastogenesis, motifs 2 and 3 are more potent than motif 1. Accordingly, inactivation of motifs 2 and 3 blocksTNF/IL-1-mediated osteoclastogenesis. Mechanistically, double mutation of motifs 2 and 3, similar to inactivation of the IVVY motif, abrogates the expression of nuclear factor of activated T-cells c1 and osteoclast genes in assays reflecting RANK-initiated and TNF/IL-1-mediated osteoclastogenesis. In contrast, double inactivation of motifs 2 and 3 did not affect the ability of RANK to activate the NF-κB and MAPK signaling pathways. Collectively, these results indicate that the RANK IVVY motif cooperates with the TRAF-binding motifs to promote osteoclastogenesis, which provides novel insights into the molecular mechanism of RANK signaling in osteoclastogenesis.