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Runt‐related transcription factor 2 (Runx2) is a key regulator of osteoblast differentiation and bone formation. In Runx2‐deficient embryos, skeletal development ceases at the cartilage anlage stage. These embryos die of respiratory failure upon birth and display a complete absence of bone and cartilage mineralization. Here, we identified Hakai, a type of E3 ubiquitin ligase as a potential Runx2 interacting partner through affinity pulldown‐based proteomic approach. Subsequently, we observed that similar to Runx2, Hakai was downregulated in osteopenic ovariectomized rats, suggesting its involvement in bone formation. Consistent with this observation, Hakai overexpression significantly enhanced osteoblast differentiation in mesenchyme‐like C3H10T1/2 as well as primary rat calvaria osteoblast (RCO) cells in vitro. Conversely, overexpression of a catalytically inactive Hakai mutant (C109A) exhibited minimal to no effect, whereas Hakai depletion markedly reduced endogenous Runx2 levels and impaired osteogenic differentiation in both C3H10T1/2 and RCOs. Mechanistically, Hakai physically interacts with Runx2 and enhances its protein turnover by rescuing it from Smad ubiquitination regulatory factor 2 (Smurf2)‐mediated proteasome degradation. Wild‐type Hakai but not Hakai‐C109A inhibited Smurf2 protein levels through proteasome‐mediated degradation. These findings underscore Hakai's functional role in bone formation, primarily through its positive modulation of Runx2 protein turnover by protecting it from Smurf2‐mediated ubiquitin‐proteasomal degradation. Collectively, our results demonstrate Hakai as a promising novel therapeutic target for osteoporosis. [ABSTRACT FROM AUTHOR]

Nitroimidazoles comprise a class of broad-spectrum anti-microbial drugs with efficacy against parasites, mycobacteria, and anaerobic Gram-positive and Gram-negative bacteria. Among these drugs, metronidazole (MTZ) is commonly used with other antibiotics to prevent infection in open fractures. However, the effect of MTZ on bone remains understudied. In this paper, we evaluated six nitroimidazole drugs for their impact on osteoblast differentiation and identified MTZ as having the highest osteogenic effect. MTZ enhanced bone regeneration at the femur osteotomy site in osteopenic ovariectomized (OVX) rats at the human equivalent dose. Moreover, in OVX rats, MTZ significantly improved bone mass and strength and improved microarchitecture compared to the vehicle-treated rats, which was likely achieved by an osteogenic mechanism attributed to the stimulation of the Wnt pathway in osteoblasts. To mitigate the reported neurological and genotoxic effects of MTZ, we designed an injectable sustained-release in situ gel formulation of the drug that improved fracture healing efficacy by 3.5-fold compared to oral administration. This enhanced potency was achieved through a significant increase in the circulating half-life and bioavailability of MTZ. We conclude that MTZ exhibits osteogenic effects, further accentuated by our sustained-release delivery system, which holds promise for enhancing bone regeneration in open fractures. [ABSTRACT FROM AUTHOR]

E3 ubiquitin ligase, ring finger protein 138 (RNF138) is involved in several biological processes; however, its role in myeloid differentiation or tumorigenesis remains unclear. RNAseq data from TNMplot showed that RNF138 mRNA levels are highly elevated in acute myeloid leukemia (AML) bone marrow samples as compared with bone marrow of normal volunteers. Here, we show that RNF138 serves as an E3 ligase for the tumor suppressor CCAAT/enhancer binding protein (C/EBPα) and promotes its degradation leading to myeloid differentiation arrest in AML. Wild-type RNF138 physically interacts with C/EBPα and promotes its ubiquitin-dependent proteasome degradation while a mutant RNF-138 deficient in ligase activity though interacts with C/EBPα, fails to down-regulate it. We show that RNF138 depletion enhances endogenous C/EBPα levels in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. Our data further shows that RNF138-mediated degradation of C/EBPα negatively affects its transactivation potential on its target genes. Furthermore, RNF138 overexpression inhibits all-transretinoic acid-induced differentiation of HL-60 cells whereas RNF138 RNAi enhances. In line with RNF138 inhibiting C/EBPα protein turnover, we also observed that RNF138 overexpression inhibited β-estradiol (E2)-induced C/EBPα driven granulocytic differentiation in C/EBPα inducible K562-p42C/EBPα-estrogen receptor cells. Furthermore, we also recapitulated these findings in PBMCs isolated from AML patients where depletion of RNF138 increased the expression of myeloid differentiation marker CD11b. These results suggest that RNF138 inhibits myeloid differentiation by targeting C/EBPα for proteasomal degradation and may provide a plausible mechanism for loss of C/EBPα expression often observed in myeloid leukemia. Also, targeting RNF138 may resolve differentiation arrest by restoring C/EBPα expression in AML. [ABSTRACT FROM AUTHOR]

A few ubiquitin ligases have been shown to target Runx2, the key osteogenic transcription factor and thereby regulate bone formation. The regulation of Runx2 expression and function are controlled both at the transcriptional and posttranslational levels. Really interesting new gene (RING) finger ubiquitin ligases of which RNF138 is a member are important players in the ubiquitin‐proteasome system, contributing to the regulation of protein turnover and cellular processes. Here, we demonstrated that RNF138 negatively correlated with Runx2 protein levels in osteopenic ovariectomized rats which implied its role in bone loss. Accordingly, RNF138 overexpression potently inhibited osteoblast differentiation of mesenchyme‐like C3H10T1/2 as well primary rat calvarial osteoblast (RCO) cells in vitro, whereas overexpression of catalytically inactive mutant RNF138Δ18‐58 (lacks RING finger domain) had mild to no effect. Contrarily, RNF138 depletion copiously enhanced endogenous Runx2 levels and augmented osteogenic differentiation of C3H10T1/2 as well as RCOs. Mechanistically, RNF138 physically associates within multiple regions of Runx2 and ubiquitinates it leading to its reduced protein stability in a proteasome‐dependent manner. Moreover, catalytically active RNF138 destabilized Runx2 which resulted in inhibition of its transactivation potential and physiological function of promoting osteoblast differentiation leading to bone loss. These findings underscore the functional involvement of RNF138 in bone formation which is primarily achieved through its modulation of Runx2 by stimulating ubiquitin‐mediated proteasomal degradation. Thus, our findings indicate that RNF138 could be a promising novel target for therapeutic intervention in postmenopausal osteoporosis. [ABSTRACT FROM AUTHOR]

Imparting epithelial to mesenchymal transition (EMT) during cellular transformation, a major driving force behind tumor progression, is one of the notorious oncogenic activities of transforming growth factor β (TGFβ); however, the secretary factors released during TGFβ‐induced EMT that may have role in potentiating EMT and tumor progression are poorly known. This study was undertaken to identify such secreted protein factors from TGFβ‐induced A549 cells cultured in serum‐free chemically defined medium (FreestyleTM) using Matrix Assisted Laser Desorption Ionization‐Time of flight/Time of flight (MALDI‐TOF/TOF) mass spectrometry. We identified some of the potential factors such as ESR, ANXA2, ALDH1A, TGFβ‐induced protein ig‐h3, and PAI‐1 that were not only secreted but some were also elevated in TGFβ‐induced A549 cells. Interestingly, these factors are widely reported to play crucial role in EMT induction and progression, which not only validates our findings but also opens avenues for further investigation, if upon secretion they act exogenously through certain receptors to potentiate cellular signaling involved in EMT induction and tumor progression. [ABSTRACT FROM AUTHOR]

CCAAT/enhancer‐binding protein α (C/EBPα), a key myeloid transcription factor, drives myeloid differentiation from blast cells by regulating the expression of granulocyte colony stimulating factor receptor and C/EBPε as required for promoting granulocyte differentiation. Here, we show that serine/threonine‐protein kinase NLK, also known as Nemo‐like kinase, physically associates with C/EBPα and phosphorylates it at multiple sites, including Ser21, Thr226, Thr230 and S234, leading to its ubiquitin‐mediated degradation. Individual phospho‐point mutants of C/EBPα could be phosphorylated by NLK, but a mutant with all phosphorylatable residues replaced by alanine resisted phosphorylation and degradation by NLK, as did the single point mutants. Furthermore, although ectopic expression of NLK enhanced phosphorylation of C/EBPα levels, it markedly inhibited total C/EBPα protein levels. Conversely, NLK depletion inhibited endogenous C/EBPα phosphorylation but enhanced its total protein levels in several acute myeloid leukemia (AML) cell lines and in peripheral blood mononuclear cells isolated from number of AML patient samples. Importantly, NLK depletion in peripheral blood mononuclear cells from primary AML patients not only restored C/EBPα protein levels, but also induced myeloid differentiation, suggesting that NLK could be therapeutically targeted to restore C/EBPα to resolve differentiation arrest in AML. [ABSTRACT FROM AUTHOR]

Context: Resuscitation of critically ill patients requires an accurate assessment of the patient's intravascular volume status. Passive leg raise cause auto transfusion of fluid to the thoracic cavity. Aims: This study aims to assess and compare the efficacy of superior vena cava (SVC) and inferior vena cava (IVC) diameter changes in response to passive leg raise (PLR) in predicting fluid responsiveness in mechanically ventilated hemodynamically unstable critically ill patients. Methods: We enrolled 30 patients. Predictive indices were obtained by transesophageal and transthoracic echocardiography and were calculated as follows: (Dmax − Dmin)/Dmax for collapsibility index of SVC (cSVC) and (Dmax − Dmin)/Dmin for distensibility index of IVC (dIVC), where Dmax and Dmin are the maximal and minimal diameters of SVC and IVC. Measurements were performed at baseline and 1 min after PLR. Patients were divided into responders (increase in cardiac index (CI) ≥10%) and nonresponders (NR) (increase in CI <10% or no increase in CI). Results: Among those included, 24 (80%) patients were R and six were NR. There was significant rise in mean arterial pressure, decrease in heart rate, and decrease in mean cSVC from baseline to 1 min after PLR among responders. The best threshold values for discriminating R from NR was 35% for cSVC, with sensitivity and specificity of being 100%, and 25% for dIVC, with 54% sensitivity and 86.7% specificity. The areas under the receiver operating characteristic curves for cSVC and dIVC regarding the assessment of fluid responsiveness were 1.00 and 0.66, respectively. Conclusions: cSVC had better sensitivity and specificity than dIVC in predicting fluid responsiveness. [ABSTRACT FROM AUTHOR]

Glycogen synthase kinase 3β (GSK3β), an ubiquitously expressed serine/threonine kinase is reported to be overexpressed and hyperactivated in cancers including acute myeloid leukemia (AML) where it promotes self-renewal, growth, and survival of AML cells. Therefore, GSK3β inhibition results in AML cell growth inhibition and myeloid differentiation. Here we identified master transcription factor PU.1 of monocyte-macrophage differentiation pathway as potential GSK3β target. We demonstrate that GSK3β phosphorylates PU.1 at Ser41 and Ser140 leading to its recognition and subsequent ubiquitin-mediated degradation by E3 ubiquitin ligase FBW7. This GSK3-dependent degradation of PU.1 by FBW7 inhibited monocyte-macrophage differentiation. We further showed that a phospho-deficient PU.1 mutant (PU.1-S41, S140A) neither bound to FBW7 nor was degraded by it. Consequently, PU.1-S41, S140A retained its transactivation, DNA-binding ability and promoted monocyte-macrophage differentiation of U937 cells even without phorbol 12-myristate 13-acetate (PMA) treatment. We further showed that FBW7 overexpression inhibited both PMA as well as M-CSF-induced macrophage differentiation of myeloid cell lines and peripheral blood mononuclear cells (PBMC) from healthy volunteers, respectively. Contrarily, FBW7 depletion promoted differentiation of these cells even without any inducer suggesting for a robust role of GSK3β-FBW7 axis in negatively regulating myeloid differentiation. Furthermore, we also recapitulated these findings in PBMCs isolated from patients with leukemia where FBW7 overexpression markedly inhibited endogenous PU.1 protein levels. In addition, PBMCs also showed enhanced differentiation when treated with M-CSF and GSK3 inhibitor (SB216763) together compared with M-CSF treatment alone. IMPLICATIONS: Our data demonstrate a plausible mechanism behind PU.1 restoration and induction of myeloid differentiation upon GSK3β inhibition and further substantiates potential of GSK3β as a therapeutic target in AML., (©2020 American Association for Cancer Research.)