RING finger E3 ligase, RNF138 inhibits osteoblast differentiation by negatively regulating Runx2 protein turnover.

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.

The Role of Osteogenic Effect and Vascular Function in Bone Health in Hypertensive Rats: A Study of Anti-hypertensive and Hemorheologic Drugs.

Vascular dysfunction contributes to the development of osteopenia in hypertensive patients, as decreased blood supply to bones results in tissue damage and dysfunction. The effect of anti-hypertensive medicines on bone mass in hypertensive individuals is inconclusive because of the varied mechanism of their action, and suggests that reducing blood pressure (BP) alone is insufficient to enhance bone mass in hypertension. Pentoxifylline (PTX), a hemorheological drug, improves blood flow by reducing blood viscosity and angiogenesis, also has an osteogenic effect. We hypothesized that improving vascular function is critical to increasing bone mass in hypertension. To test this, we screened various anti-hypertensive drugs for their in vitro osteogenic effect, from which timolol and hydralazine were selected. In adult female spontaneously hypertensive rats (SHRs), timolol and hydralazine did not improve vascular function and bone mass, but PTX improved both. In female SHR animals, PTX restored bone mass, strength and mineralization, up to the level of normotensive control rats. In addition, we observed lower blood vasculature in the femur of adult SHR animals, and PTX restored them. PTX also restored the bone vascular and angiogenesis parameters that had been impaired in OVX SHR compared to sham SHR. This study demonstrates the importance of vascular function in addition to increased bone mass for improving bone health as achieved by PTX without affecting BP, and suggests a promising treatment option for osteoporosis in hypertensive patients, particularly at-risk postmenopausal women.

Exome sequencing identified mutations in the WNT1 and COL1A2 genes in osteogenesis imperfecta cases.

BACKGROUND: Osteogenesis imperfecta (OI) is a heritable connective tissue disorder characterized by bone deformities, fractures and reduced bone mass. OI can be inherited as a dominant, recessive, or X-linked disorder. The mutational spectrum has shown that autosomal dominant mutations in the type I collagen-encoding genes are responsible for OI in 85% of the cases. Apart from collagen genes, mutations in more than 20 other genes, such as CRTAP, CREB3L1, MBTPS2, P4HB, SEC24D, SPARC, FKBP10, LEPRE1, PLOD2, PPIB, SERPINF1, SERPINH1, SP7, WNT1, BMP1, TMEM38B, and IFITM5 have been reported in OI. METHODS AND RESULTS: To understand the genetic cause of OI in four cases, we conducted whole exome sequencing, followed by Sanger sequencing. In case #1, we identified a novel c.506delG homozygous mutation in the WNT1 gene, resulting in a frameshift and early truncation of the protein at the 197th amino acid. In cases #2, 3 and 4, we identified a heterozygous c.838G > A mutation in the COL1A2 gene, resulting in a p.Gly280Ser substitution. The clinvar frequency of this mutation is 0.000008 (GnomAD-exomes). This mutation has been identified by other studies as well and appears to be a mutational hot spot. These pathogenic mutations were found to be absent in 96 control samples analyzed for these sites. The presence of these mutations in the cases, their absence in controls, their absence or very low frequency in general population, and their evaluation using various in silico prediction tools suggested their pathogenic nature. CONCLUSIONS: Mutations in the WNT1 and COL1A2 genes explain these cases of osteogenesis imperfecta.

Small secretory proteins of immune cells can modulate gynecological cancers.

Small secretory proteins of immune cells are mostly Cytokines, which include chemokines, interleukins, interferons, lymphokines and tumor necrosis factors but not hormones or growth factors. These secretory proteins are the molecular messengers and primarily involved in autocrine, paracrine and endocrine signaling as immunomodulating agents. Hence, these proteins actually regulate the cells of immune system to communicate with one another to produce a synchronized, robust, still self-regulated response to a specific antigen. Chemokines are smaller secreted proteins that control overall immune cell movement and location; these chemokines are divided into 4 subgroups, namely, CXC, CC, CX3C and C according to the position of 4 conserved cysteine residues. Complete characterization of cytokines and chemokines can exploit their vast signaling networks to develop cancer treatments. These secretory proteins like IL-6, IL-10, IL-12, TNFα, CCL2, CXCL4 & CXCL8 are predominantly expressed in most of the gynecological cancers, which directly stimulate immune effector cells and stromal cells at the tumor site and augment tumor cell recognition by cytotoxic T-cells. Hence; these secretory proteins are the major regulators, which can actually modulate all kinds of gynecological cancers. Furthermore, advancements in adoptive T-cell treatment have relied on the use of multiple cytokines/chemokines to establish a highly regulated environment for anti-tumor T cell growth. A number of in vitro studies as well as animal models and clinical subjects have also shown that cytokines/chemokines have broad antitumor activity, which has been translated into a number of cancer therapy approaches. This review will focus on the foremost cytokines & chemokines involved in the majority of the gynecological malignancies and discuss their basic biology as well as clinical applications.

Multifaceted role of chemokines in solid tumors: From biology to therapy.

Chemokines are small secretory chemotactic cytokines that control the directed migration of immune cells. Chemokines are involved in both anti-and pro-tumorigenic immune responses. Accumulating evidence suggests that the balance between these responses is influenced by several factors such as the stage of tumorigenesis, immune cell activation, recruitment of immune activating or immunosuppressive cells in the tumor microenvironment (TME), and chemokine receptor expression on effector and regulatory target cells. Cancer cells engage in a complex network with their TME components via several factors including growth factors, cytokines and chemokines that are critical for the growth of primary tumor and metastasis. However, chemokines show a multifaceted role in tumor progression including maintenance of stem-like properties, tumor cell proliferation/survival/senescence, angiogenesis, and metastasis. The heterogeneity of solid tumors in primary and metastatic cancers presents a challenge to the development of successful cancer therapy. Despite extensive research on how solid tumors escape immune cell-mediated anti-tumor response, finding an effective therapy for metastatic cancer still remains a challenge. This review discusses the multifarious roles of chemokines in solid tumors including various chemokine signaling pathways such as CXCL8-CXCR1/2, CXCL9, 10, 11-CXCR3, CXCR4-CXCL12, CCL(X)-CCR(X) in primary and metastatic cancers. We further discuss the novel therapeutic approaches that have been developed by major breakthroughs in chemokine research to treat cancer patients by the strategic blockade/activation of these signaling axes alone or in combination with immunotherapies.

Injectable Bone Cement Reinforced with Gold Nanodots Decorated rGO-Hydroxyapatite Nanocomposites, Augment Bone Regeneration.

Interest in the development of new generation injectable bone cements having appropriate mechanical properties, biodegradability, and bioactivity has been rekindled with the advent of nanoscience. Injectable bone cements made with calcium sulfate (CS) are of significant interest, owing to its compatibility and optimal self-setting property. Its rapid resorption rate, lack of bioactivity, and poor mechanical strength serve as a deterrent for its wide application. Herein, a significantly improved CS-based injectable bone cement (modified calcium sulfate termed as CSmod ), reinforced with various concentrations (0-15%) of a conductive nanocomposite containing gold nanodots and nanohydroxyapatite decorated reduced graphene oxide (rGO) sheets (AuHp@rGO), and functionalized with vancomycin, is presented. The piezo-responsive cement exhibits favorable injectability and setting times, along with improved mechanical properties. The antimicrobial, osteoinductive, and osteoconductive properties of the CSmod cement are confirmed using appropriate in vitro studies. There is an upregulation of the paracrine signaling mediated crosstalk between mesenchymal stem cells and human umbilical vein endothelial cells seeded on these cements. The ability of CSmod to induce endothelial cell recruitment and augment bone regeneration is evidenced in relevant rat models. The results imply that the multipronged activity exhibited by the novel-CSmod cement would be beneficial for bone repair.

Efficient Nitric Oxide Scavenging by Urea-Functionalized Push-Pull Chromophore Modulates NO-Mediated Diseases.

The excess nitric oxide (NO) produced in the body in response to bacterial/proinflammatory stimuli is responsible for several pathological conditions. The current approaches that target the production of excess NO, either through the inhibition of nitric oxide synthase enzyme or its downstream mediators have been clinically unsuccessful. With an aim to regulate the excess NO, urea-functionalized push-pull chromophores containing 1,1,4,4-tetracyanobuta-1,3-dienes (TCBD) or expanded TCBD (eTCBD) were developed as NO scavengers. The NMR mechanistic studies revealed that upon NO binding, these molecules are converted to uncommon stable NONOates. The unique emissive property of Urea-eTCBD enables its application in vitro, as a NO-sensor. Furthermore, the cytocompatible Urea-eTCBD, rapidly inactivated the NO released from LPS-activated cells. The therapeutic efficacy of the molecule in modulating NO-mediated pathological condition was confirmed using a carrageenan-induced inflammatory paw model and a corneal injury model. While the results confirm the advantages of scavenging the excess NO to address a multitude of NO-mediated diseases, the promising sensing and bioactivity of Urea-eTCBD can motivate further exploration of such molecules in allied areas of research.

Post-Implantation Stiffening by a Bioinspired, Double-Network, Self-Healing Hydrogel Facilitates Minimally Invasive Cell Delivery for Cartilage Regeneration.

Injectable hydrogels have demonstrated advantages in cartilage repair by enabling the delivery of cells through a minimally invasive approach. However, several injectable hydrogels suffer from rapid degradation and low mechanical strength. Moreover, higher mechanical stiffness in hydrogels can have a detrimental effect on post-implantation cell viability. To address these challenges, we developed an in situ forming bioinspired double network hydrogel (BDNH) that exhibits temperature-dependent stiffening after implantation. The BDNH mimics the microarchitecture of aggrecan, with hyaluronic acid-conjugated poly(N-isopropylacrylamide) providing rigidity and Schiff base crosslinked polymers serving as the ductile counterpart. BDNHs exhibited self-healing property and enhanced stiffness at physiological temperature. Excellent cell viability, long time cell proliferation, and cartilage specific matrix production were observed in the chondrocytes cultured in the BDNH hydrogel. Evidence of cartilage regeneration in a rabbit cartilage defect model using chondrocyte-laden BDNH has suggested it to be a potential candidate for cartilage tissue engineering.

Proposing a clinical algorithm for better diagnosis of hypophosphatasia in resource-limiting situations.

Early diagnosis of hypophosphatasia (HPP) is challenging. Here, we propose to broaden the diagnostic criteria of HPP by reviewing published data on BMD and fractures in HPP patients. Non-osteoporotic fractures and higher than normal lumbar BMD were recurrent in HPP patients and could be included as diagnostic criteria. HPP is a genetic disorder caused by autosomal recessive or dominant loss-of-function mutations in the ALPL gene that encodes for tissue-nonspecific alkaline phosphatase (TNSALP). Expressive genetic heterogeneity and varying severity of TNSALP deficiency lead to a wide-ranging presentation of skeletal diseases at different ages that coupled with HPP's rarity and limitation of biochemical and mutational studies present serious hurdles to early diagnosis and management of HPP. To widen the scope of HPP diagnosis, we assessed the possibility of areal bone mineral density (BMD) as an additional clinical feature of this disease. PubMed, Web of Science, and ScienceDirect were searched with the following keywords: ("Hypophosphatasia OR HPP") AND ("Bone Mineral Density OR BMD") AND "Human". Studies and case reports of subjects with age ≥ 18 years and having BMD data were included. We pooled data from 25 publications comprising 356 subjects (90 males, 266 females). Only four studies had a control group. Biochemical hallmarks, pyridoxal 5'-phosphate (PLP) and phosphoethanolamine (PEA), were reported in fifteen and six studies, respectively. Twenty studies reported genetic data, nineteen studies reported non-vertebral fractures, all studies reported lumbar spine (LS) BMD, and nineteen reported non-vertebral BMD. Higher than normal and normal BMD at LS were reported in three and two studies, respectively. There was marked heterogeneity in BMD at the non-vertebral sites. Higher than normal or normal LS BMD in an adult with minimal or insufficient fractures, pseudofractures, non-healing fractures, fragility fractures, and stress fractures may be included in the diagnostic protocol of HPP. However, genetic testing is recommended for a definitive diagnosis.

Oral Administration of Isovitexin, a Naturally Occurring Apigenin Derivative Showed Osteoanabolic Effect in Ovariectomized Mice: A Comparative Study with Teriparatide.

Isovitexin (apigenin-6C-glucopyranose) is found in several food items and medicinal plants. Recently, we showed that isovitexin stimulated osteoblast differentiation through mitochondrial biogenesis and respiration that required adiponectin receptors (AdipoRs). Here, we studied whether oral isovitexin has a bone anabolic effect in vivo. At first, using a femur osteotomy model in adult mice, we compared the bone regenerative effect of isovitexin and apigenin. Whereas isovitexin-stimulated bone formation at the osteotomy site at 2.5 mg/kg and 5 mg/kg dose, apigenin had no effect. Subsequently, we tested the effect of isovitexin (5 mg/kg) in ovariectomized (OVX) osteopenic mice and observed that it restored bone mass and architecture of trabecular bones (femur metaphysis and fifth lumbar vertebra/L5) and cortical bones (femur diaphysis). Isovitexin completely restored bone strength at L5 (compressive strength) and femur (bending strength) in OVX mice. The bone anabolic effect of isovitexin was demonstrated by the increased surface referent bone formation parameters, increased expression of osteogenic genes (Runx2, bone morphogenetic protein-2 and type 1 collagen) in bones, and increased serum procollagen type 1N-terminal propeptide in OVX mice and these were on a par with teriparatide. Isovitexin inhibited bone and serum sclerostin as well as the serum type I collagen cross-linked C-telopeptide in OVX mice. Isovitexin has an oral bioavailability of 14.58%. Taken together, our data show that isovitexin had a significant oral bioavailability that translated to osteoanabolic effect equivalent to teriparatide and inhibited bone resorption, which implied a durable effect over teriparatide.

Bone Mineral Density, Bone Microarchitecture and Vertebral Fractures in Male Patients with Alcohol Use Disorders.

AIM: To investigate abnormalities in bone mineral density, trabecular bone score and vertebral fractures in male patients with alcohol use disorder to understand the impact on bone health. METHODS: The study subjects included 134 male patients. Controls were 134 age matched healthy males. Assessments were made of the bone mineral density (BMD), trabecular bone score (TBS) and vertebral morphometry (VFA) for vertebral fractures. Biochemical measurements included serum total T4, thyroid stimulating hormone (TSH), parathyroid hormone (PTH) and 25- Hydroxyvitamin D 25(OH) D. RESULTS: The mean BMD at total forearm, proximal forearm (or distal 1/3) and mid forearm was significantly higher in the alcohol use disorders (AUD) group than the controls (P < 0.01). Around 15% of patients with AUD had VFs compared with 9.0% of the healthy controls (P = 0.19). For each kg/m2 gain in body mass index (BMI), lumbar spine and total hip BMD increased by 0.009 and 0.014 g/cm2, respectively. Lumbar and hip BMD decreased by 0.002 and 0.003 g/cm2 per year increase in duration of alcohol used. For every 5 years increase in age of the patients the odds of having VFs increased by 39% (odds ratio 1.393 [95% confidence interval = 1.031-1.881, P = 0.03]). CONCLUSION: The findings of the current study suggest that persons with AUD in third and fourth decades of life, with BMI in normal range and with alcohol use disorder duration of around one decade might have no major alteration in BMD and TBS. Impact of alcohol use in this population was manifest by marginal increase in the prevalence of mild grade of vertebral fractures, mostly in the thoracic region.

Genistein lowers fertility with pronounced effect in males: Meta-analyses on pre-clinical studies.

Genistein, an isoflavonoid, is found in a plethora of plant-based foods, and has been approved for use in various therapies. A couple of studies in adult men observed a negative correlation between genistein exposure and reproductive parameters. To assess the effects of genistein exposure on reproduction and fertility in males and females, we performed quantitative meta-analyses by pooling data from published studies on animals that assessed various reproductive parameters. Pooled analysis showed significant decreases in sperm count in males exposed to genistein during adulthood (Hedges's g = -2.51, p = 0.013) and in utero (Hedges's g = -0.861, p = 0.016) compared with controls. In males exposed to genistein in utero, serum testosterone levels decreased (Hedges's g = -6.301, p = 0.000) and luteinizing hormone (LH) (Hedges's g = 7.127, p = 0.000) and FSH (Hedges's g = 6.19, p = 0.000) levels increased in comparison with controls. In females, the number of corpora lutea (Hedges's g = -2.103, p = 0.019) and the litter size (Hedges's g = -1.773, p-value = 0.000) decreased; however, female reproductive hormones remained unaffected. These meta-analyses show that genistein has detrimental effects on male reproductive system and on the progression and sustenance of pregnancy, with more pronounced adverse impact in males, particularly when exposed in utero.

Human Relevance of Preclinical Studies on the Skeletal Impact of Inflammatory Bowel Disease: A Systematic Review and Meta-Analysis.

Inflammatory bowel disease (IBD) is a relapsing chronic idiopathic inflammatory condition. The increased risks of fractures in the spine and decreased BMD at all weight-bearing skeletal sites have been reported in IBD patients. The understanding of the mechanisms of IBD-induced bone loss is far from complete. Appropriate animal models are a prerequisite for studying IBD-induced bone loss, which prompted us to undertake quantitative meta-analyses by pooling data from the available IBD models that assessed various bone parameters. Sufficient data for meta-analysis are obtained from chemically- but not genetically induced models. Among the chemically induced models, only the effects of dextran sulfate sodium (DSS) and 2,4,6-trinitrobenzene sulfonic acid (TNBS) on bone parameters have been reported. Meta-analysis showed that both DSS (Hedge's g = 2.124, p = 0.001) and TNBS (Hedge's g = 6.292, p = 0.000) increased inflammatory disease severity. In pooled analysis, bone volumes in femur (Hedge's g = - 3.42, p = 0.000) and tibia (Hedge's g = - 2.49, p = 0.000) showed significant losses upon DSS administration. Similarly, bone formation rate was significantly reduced upon IBD induction (Hedge's g = - 3.495, p = 0.006). Besides, cortical thickness was reduced and trabecular microstructure deteriorated by IBD induction. Insufficient data precluded us from determining the effect of IBD on bone strength and calciotropic hormones, as well as the impact of proinflammatory cytokines on bone turnover. This meta-analysis showed that IBD induction in rodents causes significant bone loss. Impaired osteoblast function appears to be the cause of this impact.

Fasciola helminth defense molecule-1 protects against experimental arthritis by inhibiting osteoclast formation and function without modulating the systemic immune response.

An inverse correlation between helminth infection and the autoimmune disease appears to be contributed by the anti-inflammatory factors produced by these organisms. Suppressing osteoclast function without affecting the systemic immunological response is an emerging therapeutic strategy for rheumatoid arthritis (RA). We observed that a synthetic peptide corresponding to 34 amino acids of C-terminal sequence of Fasciola helminth defense molecule-1 (C-FhHDM-1) inhibited RANKL-induced osteoclast formation and lysosomal acidification with an attendant upregulation of sequestome1/p62, a negative regulator of NF-κB expression. C-FhHDM-1 also suppressed RANKL production from osteoblasts. Macrophages are the major inflammatory cells in the joints of RA and C-FhHDM-1 suppressed ICAM-1 (an inflammatory surrogate) expression in these cells. In a murine model of collagen II-induced arthritis (CIA), C-FhHDM-1 improved clinical score, protected against cartilage destruction, and maintained bone mass and bone architecture of joints compared with the CIA group. C-FhHDM-1 suppressed the CIA-induced expression of TNF, IL-17, and IFN-γ in joints but not their serum levels. The peptide also had no effect on the CIA-induced suppression of T regulatory response. We conclude that C-FhHDM-1 has a joint-specific protective effect in experimental arthritis without mitigating systemic inflammation, and thus could become an adjuvant anti-arthritis therapy to prevent RA-induced osteopenia.

Regulatory safety pharmacology and toxicity assessments of a standardized stem extract of Cassia occidentalis Linn. in rodents.

Cassia occidentalis Linn (CO) is an annual/perennial plant having traditional uses in the treatments of ringworm, gastrointestinal ailments and piles, bone fracture, and wound healing. Previously, we confirmed the medicinal use of the stem extract (ethanolic) of CO (henceforth CSE) in fracture healing at 250 mg/kg dose in rats and described an osteogenic mode of action of four phytochemicals present in CSE. Here we studied CSE's preclinical safety and toxicity. CSE prepared as per regulations of Current Good Manufacturing Practice for human pharmaceuticals/phytopharmaceuticals and all studies were performed in rodents in a GLP-accredited facility. In acute dose toxicity as per New Drug and Clinical Trial Rules, 2019 (prior name schedule Y), in rats and mice and ten-day dose range-finding study in rats, CSE showed no mortality and no gross abnormality at 2500 mg/kg dose. Safety Pharmacology showed no adverse effect on central nervous system, cardiovascular system, and respiratory system at 2500 mg/kg dose. CSE was not mutagenic in the Ames test and did not cause clastogenicity assessed by in vivo bone marrow genotoxicity assay. By a sub chronic (90 days) repeated dose (as per OECD, 408 guideline) study in rats, the no-observed-adverse-effect-level was found to be 2500 mg/kg assessed by clinico-biochemistry and all organs histopathology. We conclude that CSE is safe up to 10X the dose required for its osteogenic effect.

Leprosy drug clofazimine activates peroxisome proliferator-activated receptor-γ and synergizes with imatinib to inhibit chronic myeloid leukemia cells.

Leukemia stem cells contribute to drug-resistance and relapse in chronic myeloid leukemia (CML) and BCR-ABL1 inhibitor monotherapy fails to eliminate these cells, thereby necessitating alternate therapeutic strategies for patients CML. The peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone downregulates signal transducer and activator of transcription 5 (STAT5) and in combination with imatinib induces complete molecular response in imatinib-refractory patients by eroding leukemia stem cells. Thiazolidinediones such as pioglitazone are, however, associated with severe side effects. To identify alternate therapeutic strategies for CML we screened Food and Drug Administration-approved drugs in K562 cells and identified the leprosy drug clofazimine as an inhibitor of viability of these cells. Here we show that clofazimine induced apoptosis of blood mononuclear cells derived from patients with CML, with a particularly robust effect in imatinib-resistant cells. Clofazimine also induced apoptosis of CD34+38- progenitors and quiescent CD34+ cells from CML patients but not of hematopoietic progenitor cells from healthy donors. Mechanistic evaluation revealed that clofazimine, via physical interaction with PPARγ, induced nuclear factor kB-p65 proteasomal degradation, which led to sequential myeloblastoma oncoprotein and peroxiredoxin 1 downregulation and concomitant induction of reactive oxygen species-mediated apoptosis. Clofazimine also suppressed STAT5 expression and consequently downregulated stem cell maintenance factors hypoxia-inducible factor-1α and -2α and Cbp/P300 interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2). Combining imatinib with clofazimine caused a far superior synergy than that with pioglitazone, with clofazimine reducing the half maximal inhibitory concentration (IC50) of imatinib by >4 logs and remarkably eroding quiescent CD34+ cells. In a K562 xenograft study clofazimine and imatinib co-treatment showed more robust efficacy than the individual treatments. We propose clinical evaluation of clofazimine in imatinib-refractory CML.

Rosiglitazone up-regulates glial fibrillary acidic protein via HB-EGF secreted from astrocytes and neurons through PPARγ pathway and reduces apoptosis in high-fat diet-fed mice.

The anti-diabetic drug and peroxisome proliferator-activated receptor-gamma (PPARγ) agonist, rosiglitazone, alters astrocyte activation; however, its mechanism remains less-known. We hypothesized participation of epidermal growth factor receptor (EGFR), known to control astrocyte reactivity. We first detected that rosiglitazone promoted glial fibrillary acidic protein (GFAP) expression in primary astrocytes as well as the mouse cerebral cortex, associated with increased EGFR activation. Screening for EGFR ligands revealed a rosiglitazone-mediated increase of heparin-binding epidermal growth factor (HB-EGF) in astrocytes, resulting in HB-EGF release into culture medium and mouse cerebrospinal fluid too. Treatment with HB-EGF-siRNA and EGFR inhibitors showed that the rosiglitazone-induced HB-EGF and p-EFGR were interdependent, which participated in GFAP increase. Interestingly, we observed that rosiglitazone could induce cellular and secreted-HB-EGF in neurons also, contributing toward the activated EGFR-induced GFAP in astrocytes. Probing whether these effects of rosiglitazone were PPARγ-linked, revealed potential PPARγ-responsive elements within HB-EGF gene. Moreover, gel-shift, site-directed mutagenesis, chromatin-immunoprecipitation and luciferase-reporter assays demonstrated a PPARγ-dependent HB-EGF transactivation. Subsequently, we examined effects of rosiglitazone in a high-fat diet-fed diabetes mouse model, and supporting observations in the normal cortical cells, identified a rosiglitazone-induced GFAP, astrocyte and neuronal HB-EGF and secreted-HB-EGF in the cerebral cortex of diabetic mice. Moreover, assessing relevance of increased HB-EGF and GFAP revealed an anti-apoptotic role of rosiglitazone in the cerebral cortex, supported by a GFAP-siRNA as well as HB-EGF-siRNA-mediated increase in cleaved-caspase 3 and 9 levels in the rosiglitazone-treated astrocyte-neuron coculture. Overall, our study indicates that rosiglitazone may protect the brain, via a PPARγ-dependent HB-EGF/EGFR signaling and increased GFAP.

M2 polarization of macrophages by Oncostatin M in hypoxic tumor microenvironment is mediated by mTORC2 and promotes tumor growth and metastasis.

Oncostatin M (OSM), an inflammatory cytokine belonging to the interleukin-6 (IL-6) superfamily, plays a vital role in multitude of physiological and pathological processes. Its role in breast tumor progression and metastasis to distant organs is well documented. Recent reports implicate OSM in macrophage M2 polarization, a key pro-tumoral phenomenon. M2 polarization of macrophages is believed to promote tumor progression by potentiating metastasis and angiogenesis. In the current study, we delineated the mechanism underlying OSM induced macrophage M2 polarization. The findings revealed that OSM skews macrophages towards an M2 polarized phenotype via mTOR signaling complex 2 (mTORC2). mTORC2 relays signals through two effector kinases i.e. PKC-α and Akt. Our results indicated that mTORC2 mediated M2 polarization of macrophages is not dependent on PKC-α and is primarily affected via Akt, particularly Akt1. In vivo studies conducted on 4T1/BALB/c mouse orthotropic model of breast cancer further corroborated these observations wherein i.v. reintroduction of mTORC2 abrogated monocytes into orthotropic mouse model resulted in diminished acquisition of M2 specific attributes by tumor associated macrophages. Metastasis to distant organs like lung, liver and bone was reduced as evident by decrease in formation of focal metastatic lesions in mTORC2 abrogated monocytes mice. Our study pinpoints key role of mTORC2-Akt1 axis in OSM induced macrophage polarization and suggests for possible usage of Oncostatin-M blockade and/or selective mTORC2 inhibition as a potential anti-cancer strategy particularly with reference to metastasis of breast cancer to distant organs such as lung, liver and bone.

Reversal of Osteopenia in Ovariectomized Rats by Pentoxifylline: Evidence of Osteogenic and Osteo-Angiogenic Roles of the Drug.

Pentoxifylline (PTX) is a non-selective phosphodiesterase inhibitor and is used for the management of intermittent claudication. We tested whether PTX has oral efficacy in stimulating new bone formation. Rat calvarial osteoblasts (RCO) were used to study the effect of PTX on osteoblast differentiation and angiogenesis. Pharmacokinetic and pharmacodynamic studies were carried out in rats to determine an oral dose of PTX. In ovariectomized (OVX) rats with osteopenia, the effect of PTX on various skeletal parameters was studied, and compared with teriparatide. Effect of PTX on angiogenic signaling was studied by immunoblotting and relevant pharmacologic inhibitors. Bone vascularity was measured by intravenous injection of polystyrene fluorospheres followed by in vivo imaging, and angiogenesis was studied in vitro by tubulogenesis of endothelial cells and in vivo by Matrigel plug assay. Effective concentration (EC50) of PTX in RCO was 8.2 nM and plasma PTX level was 7 nM/mL after single oral dosing of 25 mg/kg, which was 1/6th the clinically used dose. At this dose, PTX enhanced bone regeneration at femur osteotomy site and completely restored bone mass, microarchitecture, and strength in OVX rats. Furthermore, PTX increased surface referent bone formation parameters and serum bone formation marker (PINP) without affecting the resorption marker (CTX-1). PTX increased the expression of vascular endothelial growth factor and its receptor in bones and osteoblasts. PTX also increased skeletal vascularity, tubulogenesis of endothelial cells and in vivo angiogenesis. Taken together, our study suggested that PTX at 16% of adult human oral dose completely reversed osteopenia in OVX rats by osteogenic and osteo-angiogenic mechanisms.

E6AP inhibits G-CSFR turnover and functions by promoting its ubiquitin-dependent proteasome degradation.

Granulocyte colony-stimulating factor receptor (G-CSFR) plays a crucial role in regulating myeloid cell survival, proliferation, and neutrophilic granulocyte precursor cells maturation. Previously, we demonstrated that Fbw7α negatively regulates G-CSFR and its downstream signaling through ubiquitin-proteasome mediated degradation. However, whether additional ubiquitin ligases for G-CSFR exist is not known. Identifying multiple E3 ubiquitin ligases for G-CSFR shall improve our understanding of activation and subsequent attenuation of G-CSFR signaling required for differentiation and proliferation. Here, for the first time we demonstrate that E6 associated protein (E6AP), an E3 ubiquitin ligase physically associates with G-CSFR and targets it for ubiquitin-mediated proteasome degradation and thereby attenuates its functions. We further show that E6AP promoted G-CSFR degradation leads to reduced phosphorylation of signal transducer and activator of transcription 3 (STAT3) which is required for G-CSF dependent granulocytic differentiation. More importantly, our finding shows that E6AP also targets mutant form of G-SCFR (G-CSFR-T718), frequently observed in severe congenital neutropenia (SCN) patients that very often culminate to AML, however, at a quite slower rate than wild type G-CSFR. In addition, our data showed that knockdown of E6AP restores G-CSFR and its signaling thereby promoting granulocytic differentiation. Collectively, our data demonstrates that E6AP facilitates ubiquitination and subsequent degradation of G-CSFR leading to attenuation of its downstream signaling and inhibition of granulocytic differentiation.