Comparison of the Protective Effects of Nebivolol and Metoprolol against LPS-Induced Injury in H9c2 Cardiomyoblasts.

Here, we, for the first time, compared the cardioprotective effects of third-generation vasodilating beta-blocker nebivolol (Neb) and conventional beta-blocker metoprolol (Met) on LPS-induced injury in H9c2 cardiomyoblasts. Our findings denoted that Neb and Met pretreatment diminish LPS-mediated cytotoxicity and oxidative stress. Concomitantly, LPS-triggered inflammatory cytokines activation was significantly suppressed by Neb but not by Met. Pretreatment with either Neb or Met alleviated LPS-mediated mitochondrial impairment by enhancing the expression of genes related to its biogenesis such as PGC-1α, NRF1, and TFAM. On the contrary, Neb but not Met-upregulated mitochondrial fusion-related genes such as OPA, and MFN2. In summary, our findings suggest that Neb and Met treatment significantly ameliorated the LPS-induced cytotoxicity and oxidative stress. Additionally, these findings suggest that Neb but not Met significantly down-regulates LPS-induced proinflammatory factors, probably by enhancing mitochondrial biogenesis and fusion.

Transient downregulation of NR4A1 leads to impaired osteoblast differentiation through the TGF-ß pathway, and Elesclomol (STA-4783) rescues this phenotype.

Bone formation is regulated by numerous factors, such as transcription factors, cytokines, and extracellular matrix molecules. Human hormone nuclear receptors (hHNR) are a family of ligand-regulated transcription factors that are activated by steroid hormones, such as estrogen and progesterone, and various lipid-soluble signals, including retinoic acid, oxysterols, and thyroid hormone. We found that an hHNR called NR4A1 was the most highly expressed after human MSC differentiation into osteoblasts by whole-genome microarray. NR4A1 knockout decreased the osteoblastic differentiation of hMSCs in terms of ALPL expression and key marker gene expression. Whole-genome microarray analysis further confirmed the decrease in key pathways when we knocked down NR4A1. Further studies with small molecule activators identified a novel molecule called Elesclomol (STA-4783), which could activate and enhance osteoblast differentiation. Elesclomol activation of hMSCs also induced the gene expression of NR4A1 and rescued the phenotype of NR4A1 KD. In addition, Elesclomol activated the TGF-ß pathway by regulating key marker genes. In conclusion, we first identified the role of NR4A1 in osteoblast differentiation and that Elesclomol is a positive regulator of NR4A1 through activation of the TGF-ß signalling pathway.

Inhibition of GSK-3ß Enhances Osteoblast Differentiation of Human Mesenchymal Stem Cells through Wnt Signalling Overexpressing Runx2.

Small-molecule-inhibitor-based bone differentiation has been recently exploited as a novel approach to regulating osteogenesis-related signaling pathways. In this study, we identified 1-Azakenpaullone, a highly selective inhibitor of glycogen synthase kinase-3ß (GSK-3ß), as a powerful inducer of osteoblastic differentiation and mineralization of human mesenchymal stem cells (MSCs). GSK-3ß is a serine-threonine protein kinase that plays a major role in different disease development. GSK-3ß is a key regulator of Runx2 activity in osteoblastic formation. We evaluated alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin Red staining to assess the mineralization of cultured human MSCs. Gene expression profiling was assessed using an Agilent microarray platform, and bioinformatics were performed using Ingenuity Pathway Analysis software. Human MSCs treated with 1-Azakenpaullone showed higher ALP activity, increased in vitro mineralized matrix formation, and the upregulation of osteoblast-specific marker gene expression. Global gene expression profiling of 1-Azakenpaullone-treated human MSCs identified 1750 upregulated and 2171 downregulated mRNA transcripts compared to control cells. It also suggested possible changes in various signaling pathways, including Wnt, TGFß, and Hedgehog. Further bioinformatics analysis employing Ingenuity Pathway Analysis recognized significant enrichment in the 1-Azakenpaullone-treated cells of genetic networks involved in CAMP, PI3K (Complex), P38 MAPK, and HIF1A signaling and functional categories associated with connective tissue development. Our results suggest that 1-Azakenpaullone significantly induced the osteoblastic differentiation and mineralization of human MSCs mediated by the activation of Wnt signaling and the nuclear accumulation of ß-catenin, leading to the upregulation of Runx2, a key transcription factor that ultimately promotes the expression of osteoblast-specific genes. Thus, 1-Azakenpaullone could be used as an osteo-promotor factor in bone tissue engineering.

JAK2 Inhibition by Fedratinib Reduces Osteoblast Differentiation and Mineralisation of Human Mesenchymal Stem Cells.

Several signalling pathways, including the JAK/STAT signalling pathway, have been identified to regulate the differentiation of human bone marrow skeletal (mesenchymal) stem cells (hBMSCs) into bone-forming osteoblasts. Members of the JAK family mediate the intracellular signalling of various of cytokines and growth factors, leading to the regulation of cell proliferation and differentiation into bone-forming osteoblastic cells. Inhibition of JAK2 leads to decoupling of its downstream mediator, STAT3, and the subsequent inhibition of JAK/STAT signalling. However, the crucial role of JAK2 in hBMSCs biology has not been studied in detail. A JAK2 inhibitor, Fedratinib, was identified during a chemical biology screen of a small molecule library for effects on the osteoblastic differentiation of hMSC-TERT cells. Alkaline phosphatase activity and staining assays were conducted as indicators of osteoblastic differentiation, while Alizarin red staining was used as an indicator of in vitro mineralised matrix formation. Changes in gene expression were assessed using quantitative real-time polymerase chain reaction. Fedratinib exerted significant inhibitory effects on the osteoblastic differentiation of hMSC-TERT cells, as demonstrated by reduced ALP activity, in vitro mineralised matrix formation and downregulation of osteoblast-related gene expression, including ALP, ON, OC, RUNX2, OPN, and COL1A1. To identify the underlying molecular mechanisms, we examined the effects of Fedratinib on a molecular signature of several target genes known to affect hMSC-TERT differentiation into osteoblasts. Fedratinib inhibited the expression of LIF, SOCS3, RRAD, NOTCH3, TNF, COMP, THBS2, and IL6, which are associated with various signalling pathways, including TGFß signalling, insulin signalling, focal adhesion, Notch Signalling, IL-6 signalling, endochondral ossification, TNF-α, and cytokines and inflammatory response. We identified a JAK2 inhibitor (Fedratinib) as a powerful inhibitor of the osteoblastic differentiation of hMSC-TERT cells, which may be useful as a therapeutic option for treating conditions associated with ectopic bone formation or osteosclerotic metastases.

Effects of all-trans retinoic acid on the in vitro maturation of camel (Camelus dromedarius) cumulus-oocyte complexes.

All-trans retinoic acid (RA) is a metabolite of vitamin A and has pleiotropic actions on many different biological processes, including cell growth and differentiation, and is involved in different aspects of fertility and developmental biology. In the current study, we investigated the effects of RA on camel (Camelus dromedarius) cumulus-oocyte complex in vitro maturation (IVM). IVM medium was supplemented with 0, 10, 20, and 40 µM RA. Application of 20 µM RA significantly reduced the proportion of degenerated oocytes and significantly improved oocyte meiosis and first polar body extrusion compared to the control and other experimental groups. Retinoic acid significantly reduced the mRNA transcript levels of apoptosis-related genes, including BAX and P53, and reduced the BAX/BCL2 ratio. In addition, RA significantly reduced the expression of the Transforming growth factor beta (TGFß) pathway-related transcripts associated with the actin cytoskeleton, ACTA2 and TAGLN; however, RA increased TGFß expression in cumulus cells. The small molecule SB-431542 inhibits the TGFß pathway by inhibiting the activity of activin receptor-like kinases (ALK-4, ALK-5, and ALK-7); however, combined supplementation with RA during IVM compensated for the inhibitory effect of SB-431542 on cumulus expansion, oocyte meiosis I, and first polar body extrusion in activated oocytes. The current study shows the beneficial effects of RA on camel oocyte IVM and provides a model to study the multifunctional mechanisms involved in cumulus expansion and oocyte meiosis, particularly those involved in the TGFß pathway.

NR2F1 mediated down-regulation of osteoblast differentiation was rescued by bone morphogenetic protein-2 (BMP-2) in human MSC.

Endochondral ossification is the process by which long bones are formed; the process of long bone formation is regulated by numerous factors such as transcription factors, cytokines, and extracellular matrix molecules. Human hormone Nuclear receptors (hHNR) are a family of ligand-regulated transcription factors that are activated by steroid hormones, such as estrogen and progesterone, and various lipid-soluble signals, including retinoic acid, oxysterols, and thyroid hormone. Whole genome microarray data from our previous study revealed that most hHNR's are up-regulated during osteoblast differentiation in hMSCS. NR2F1 was among the highest expressed hHNR during osteogenesis, NR2F1 belongs to the steroid/thyroid hormone nuclear receptor superfamily. NR2F1 is designated as an orphan nuclear receptor because its ligands are unknown. NR2F1 plays a wide range of roles, including cell differentiation, cancer progression, and central and peripheral neurogenesis. Identifying signaling networks involved in osteoblast differentiation is important in orchestrating new therapeutic and clinical applications in bone biology. This study aimed to identify alterations in signaling networks mediated by NR2F1 in osteoblast differentiation. siRNA-mediated down-regulation of NR2F1 leads to impairment in the differentiation of hBMSC-TERT to osteoblast; gene-expression results confirmed the down-regulation of osteoblast markers such as RUNX2, ALPL, OSC, and BSP. Global whole gene expression analysis revealed that most down-regulated genes were associated with osteoblast differentiation (DDIT3, BMP2). Pathway analysis revealed prominent signaling pathways that were down-regulated, including the TGFß pathway and MAPK pathway. Functional studies on NR2F1 transfected cells, during osteoblast differentiation in combination with TGFß1 and BMP-2, showed that TGFß1 does not recover osteoblast differentiation, whereas BMP-2 rescues osteoblast differentiation in NR2F1 siRNA transfected cells. Thus, our results showed that BMP-2 could intervene in NR2F1 down-regulated signaling pathways to recover osteoblast differentiation.

Differences between the tolerance of camel oocytes and cumulus cells to acute and chronic hyperthermia.

The dromedary camel (Camel dromedarius) is physiologically well adapted to life in hot, dry and barren land. In the present study, we report the tolerance of camel oocytes and cumulus cells to acute and chronic heat shock. Camel oocytes and cumulus cells were exposed to acute (45 °C for 2 h) and chronic (45 °C for 20 h) heat shock. Our results demonstrated that acute and chronic heat shock altered malondialdehyde concentration, which is a marker for oxidative stress. Furthermore, the heat shock reduced glutathione levels during in vitro oocyte maturation. The expression of two well-known heat shock proteins HSP70 and HSP90 were increased similarly in oocytes and cumulus cells after acute heat shock. Oocytes were less tolerant to the short acute heat shock, and showed decreased maturation, which leads to reduction in ooplasmic diameter and an increase in chromosomal count abnormalities. Furthermore, the pro-apoptotic genes P53 and BAX had increased expression levels, whereas for the anti-apoptotic gene such as BCL2 expression levels was decreased. On the other hand, the cumulus cells tolerated acute and chronic heat shock, as evident by the increase in HSP70 and HSP90 expression and steady expression levels of P53, BAX, and BCL2 after acute hyperthermia. Cumulus cells regained their vitality and ability to proliferate after chronic hyperthermia and showed wound healing capabilities after 9 days of chronic hyperthermia. Collectively, these results indicate the adaptive tolerance of camel somatic cells to acute and chronic heat shock, which is lethal to cells in many other mammals.

Bone morphogenetic protein 2 (BMP2) induces growth suppression and enhances chemosensitivity of human colon cancer cells.

BACKGROUND: Molecular profiling of colorectal cancer (CRC) based on global gene expression has revealed multiple dysregulated signalling pathways associated with drug resistance and poor prognosis. However, the role of BMP2 signaling in CRC is not fully characterised. METHODS: Bioinformatics data analysis were conducted on the GSE21510 dataset. Leniviral technology was utilized to stably express BMP2 in the HCT116 CRC model. Gene expression profiling was conducted using Agilent microarray platform while data normalization and bioinformatics were conducted using GeneSpring software. Changes in gene expression were assessed using qRT-PCR. AlamarBlue assay was used to assess cell viability in vitro. In vivo experiments were conducted using SCID mice. RESULTS: Our data revealed frequent downregulation of BMP2 in primary CRC tissues. Additionally, interrogation of publically available gene expression datasets revealed significant downregulation of BMP2 in metastatic recurrent compared to non-metastatic cancer (p = 0.02). Global gene expression analysis in CRC cells over-expressing BMP2 revealed multiple dysregulated pathways mostly affecting cell cycle and DNA damage response. Concordantly, lentiviral-mediated re-expression of BMP2 inhibited HCT116 CRC growth, sphere formation, clonogenic potential, cell migration, and sensitized CRC cells to 5-fluorouracil (5-FU) in vitro. Additionally, BMP2 inhibited CRC tumor formation in SCID mice. CONCLUSIONS: Our data revealed an inhibitory role for BMP2 in CRC, suggesting that restoration of BMP2 expression could be a potential therapeutic strategy for CRC.

Deferent Anatomical Presentations of Iliolumbar Ligament: A Cadaveric Study.

This work was carried out to describe the detailed gross anatomy of the iliolumbar ligaments in human cadavers and to shed more light on these disputes regarding the configuration and direction of these ligaments. Twenty partially dissected human formalin-preserved cadavers originating from North America and Europe were investigated in this study. Blunt dissection was made through the ventral and dorsal aspects of the pelvic area of the cadavers. According to the current study, the anterior and posterior portions of the iliolumbar ligament most frequently attached to the 5th lumbar vertebra's transverse process (70% and 80%, respectively). The body of the 4th lumbar vertebra with the 5th lumbar vertebra' transverse process was the attachment of the anterior part (30%). The attachment of the posterior part was the body of the 5th lumbar vertebra (20%). The anterior and posterior parts of the iliolumbar ligament were inserted into the anterior tip of the iliac crest. There is an obvious variation in the morphological appearance of the iliolumbar ligament distinguished in attachments, length, width, thickness, number of bands, and the presence of accessory bands in the anterior part of the ligament. In addition, a new attachment for the anterior band was revealed in one-third of the specimens (body of the 4th lumbar vertebra) which have not been described before. Also, in one-fifth of the specimens, there was a new attachment for the posterior band (body of the 5th lumbar vertebra).

A New Procedure in Bone Engineering Using Induced Adipose Tissue.

Background: Osteoporosis is associated with a metabolic imbalance between adipogenesis and osteogenesis. We hypothesized that implanting a carrier for differentiated stem cells and signaling molecules inside adipose tissues could be used to enable transdifferentiation between cells, upregulate osteogenesis, and support bone formation, which may regain the balance between osteogenesis and adipogenesis. Methodology: A CL1 human mesenchymal stem cell line was grown in an osteogenic medium to differentiate into osteoblasts, and the differentiated cells were then exposed to an adipogenic medium to stimulate differentiation into adipocytes. Osteogenic and adipogenic differentiation were confirmed by the following assays: alkaline phosphatase staining, Nile red Staining, and quantitative real-time polymerase chain reaction (qPCR). The ratio of adipocytes to osteocytes for both cases was calculated. To evaluate bone induction in vivo, a calcium sulfate/hydroxyapatite cement was prepared in a syringe and then seeded with 106 cells/mL of rat bone marrow stromal cells (rMSCs) and covered with 1 mL of tissue culture media containing 0.1 mg of bone morphogenetic protein 7 (BMP-7). The construct was injected into the abdominal fat tissue of 10 male Sprague-Dawley rats. Results: The conversion of osteocytes to adipocytes was 20-fold greater than the reverse conversion, and the area of bone regeneration was 15.7 ± 3.7%, the area of adipose tissue was 65.8 ± 13.1%, and the area of fibrous tissue was 18.3 ± 7.8%. Conclusion: Adipogenic interconversion and associated bone formation demonstrate the potential of a new therapy for balancing osteogenesis and adipogenesis.

Apigenin Reverses Interleukin-1ß-Induced Suppression of Adipocyte Browning via COX2/PGE2 Signaling Pathway in Human Adipocytes.

SCOPE: Inflammatory responses to obesity, including interleukin-1 beta (IL-1ß) activation, downregulate mitochondrial function and interfere with adipocyte browning, an important component of energy expenditure. This study investigates the impact of apigenin (Apg), a natural flavonoid with anti-inflammatory properties, on adipocyte browning in the presence of IL-1ß. METHODS AND RESULTS: Apg protects dibutyryl-cAMP-induced browning from IL-1ß in primary human adipocytes, as evidenced by increased brown-specific markers, mitochondrial content, and oxygen consumption. Apg significantly represses inflammatory markers and NF-κB activation induced by IL-1ß in these adipocytes. Intriguingly, Apg profoundly induces cyclooxygenase 2 (COX2) and prostaglandin E2 (PGE2) expression in response to IL-1ß treatment. Conversely, COX2 pharmacological inhibition or RNA silencing attenuates the positive effect of Apg on adipocyte browning in IL-1ß-treated cells. Additionally, blockage of PGE2 receptor 4 (EP4) attenuates Apg-mediated adipocyte browning. The effect of Apg on adipocyte browning in IL-1ß-treated adipocytes is accompanied by an elevation in intracellular Ca2+ , partly due to TRPV1/4 receptor activation. CONCLUSION: Apg plays a protective role against inflammation-induced suppression of adipocyte browning by dampening inflammation and activating the COX2/PGE2 axis for uncoupling protein 1 induction via EP4 activation. These data unravel the novel therapeutic values of Apg for treating obesity via adipocyte browning stimulation.

Tankyrase inhibitor XAV-939 enhances osteoblastogenesis and mineralization of human skeletal (mesenchymal) stem cells.

Tankyrase is part of poly (ADP-ribose) polymerase superfamily required for numerous cellular and molecular processes. Tankyrase inhibition negatively regulates Wnt pathway. Thus, Tankyrase inhibitors have been extensively investigated for the treatment of clinical conditions associated with activated Wnt signaling such as cancer and fibrotic diseases. Moreover, Tankyrase inhibition has been recently reported to upregulate osteogenesis through the accumulation of SH3 domain-binding protein 2, an adaptor protein required for bone metabolism. In this study, we investigated the effect of Tankyrase inhibition in osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSCs). A Tankyrase inhibitor, XAV-939, identified during a functional library screening of small molecules. Alkaline phosphatase activity and Alizarin red staining were employed as markers for osteoblastic differentiation and in vitro mineralized matrix formation, respectively. Global gene expression profiling was performed using the Agilent microarray platform. XAV-939, a Tankyrase inhibitor, enhanced osteoblast differentiation of hBMSCs as evidenced by increased ALP activity, in vitro mineralized matrix formation, and upregulation of osteoblast-related gene expression. Global gene expression profiling of XAV-939-treated cells identified 847 upregulated and 614 downregulated mRNA transcripts, compared to vehicle-treated control cells. It also points towards possible changes in multiple signaling pathways, including TGFß, insulin signaling, focal adhesion, estrogen metabolism, oxidative stress, RANK-RANKL (receptor activator of nuclear factor κB ligand) signaling, Vitamin D synthesis, IL6, and cytokines and inflammatory responses. Further bioinformatic analysis, employing Ingenuity Pathway Analysis identified significant enrichment in XAV-939-treated cells of functional categories and networks involved in TNF, NFκB, and STAT signaling. We identified a Tankyrase inhibitor (XAV-939) as a powerful enhancer of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with low bone formation.

Transgelin is a poor prognostic factor associated with advanced colorectal cancer (CRC) stage promoting tumor growth and migration in a TGFß-dependent manner.

Colorectal cancer (CRC) is the fourth most common cancer type globally. Investigating the signaling pathways that maintain cancer cell phenotype can identify new biomarkers for targeted therapy. Aberrant transforming growth factor-ß (TGFß) signaling has been implicated in CRC progression, however, the exact mechanism by which TGFß exerts its function is still being unraveled. Herein, we investigated TAGLN expression, prognostic value, and its regulation by TGFß in CRC. While TAGLN was generally found to be downregulated in CRC, elevated expression of TAGLN was associated with advanced CRC stage and predicted poor overall survival (hazard ratio (HR) = 1.8, log-rank test P-value = 0.014) and disease-free survival (HR = 1.6, log-rank test P-value = 0.046), hence implicating TAGLN as poor prognostic factor in CRC. Forced expression of TAGLN was associated with enhanced CRC cell proliferation, clonogenic growth, cell migration and in vivo tumor formation in immunocompromised mice, while targeted depletion of TAGLN exhibited opposing biological effects. Global gene expression profiling of TAGLN-overexpressing or TAGLN-deficient CRC cell lines revealed deregulation of multiple cancer-related genes and signaling pathways. Transmission electron microscopy (TEM) revealed ultrastructural changes due to loss of TAGLN, including disruption of actin cytoskeleton organization and aberrant actin filament distribution. Hierarchical clustering, principle component, and ingenuity pathway analyses revealed distinct molecular profile associated with TAGLNhigh CRC patients with remarkable activation of a number of mechanistic networks, including SMARCA4, TGFß1, and P38 MAPK. The P38 MAPK was the top predicted upstream regulator network promoting cell movement through regulation of several intermediate molecules, including TGFß1. Concordantly, functional categories associated with cellular movement and angiogenesis were also enriched in TAGLNhigh CRC, supporting a model for the molecular mechanisms linking TGFß-induced upregulation of TAGLN and CRC tumor progression and suggesting TAGLN as potential prognostic marker associated with advanced CRC pathological stage.

MicroRNA Expression Profiling on Paired Primary and Lymph Node Metastatic Breast Cancer Revealed Distinct microRNA Profile Associated With LNM.

Breast cancer (BC) is the foremost cause of cancer-related deaths in women. BC patients are oftentimes presented with lymph node metastasis (LNM), which increases their risk of recurrence. Compelling data have recently implicated microRNAs in promoting BC metastasis. Therefore, the identification of microRNA (miRNA)-based molecular signature associated with LNM could provide an opportunity for a more personalized treatment for BC patients with high risk of LNM. In current study, we performed comprehensive miRNA profiling in matched primary breast and LNM and identified 40 miRNAs, which were differentially expressed in LNM compared to primary tumors. The expression of 14 miRNAs (Up: hsa-miR-155-5p, hsa-miR-150-5p, hsa-miR-146a-5p, hsa-miR-142-5p and down: hsa-miR-200a-3p, hsa-miR-200b-3p, hsa-miR-200c-3p, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-214-3p, hsa-miR-141-3p, hsa-miR-127-3p, hsa-miR-125a-5p, and hsa-let-7c-5p) was subsequently validated in a second cohort of 32 breast and 32 matched LNM tumor tissues. Mechanistically, forced expression of hsa-miR-205-5p, or hsa-miR-214-3p epigenetically inhibited MDA-MB-231 cell proliferation, colony formation, and cell migration. Global gene expression profiling on MDA-MB-231 cells overexpressing hsa-miR-205-5p, or hsa-miR-214-3p in combination with in silico target prediction and ingenuity pathway analyses identified multiple bona fide targets for hsa-miR-205-5p, hsa-miR-214-3p affecting cellular proliferation and migration. Interestingly, interrogation of the expression levels of hsa-miR-205 and hsa-miR-214 in the METABRIC breast cancer dataset revealed significantly poor overall survival in patients with downregulated expression of miR-205 [HR = 0.75 (0.61-0.91)], p = 0.003 and hsa-miR-214 [HR = 0.74 (0.59-0.93) p = 0.008]. Our data unraveled the miRNA-transcriptional landscape associated with LNM and provide novel insight on the role of several miRNAs in promoting BC LNM, and suggest their potential utilization in the clinical management of BC patients.

Thermotolerance and plasticity of camel somatic cells exposed to acute and chronic heat stress.

The Arabian camel is the largest known mammal that can survive in severe hot climatic conditions. We provide the molecular explanation for the thermotolerance of camel granulosa somatic cells after exposure to 45 °C for 2 (acute heat shock) or 20 h (chronic heat shock). The common features of the cellular responses to acute heat stress were the increase of heat shock proteins and DNA repair enzymes expression. Actin polymerization and Rho signaling were critically activated as a cellular defense against heat shock. Cells exposed to chronic heat shock showed altered cell architecture with a decrease in total detected proteins, metabolic enzymes, and cytoskeletal protein expression. Treatment with transforming growth factor beta (TGFß) pathway inhibitor SB-431542 suppressed the morphological alterations of cells exposed to chronic heat shock. Moreover, during the recovery stage at 38 °C for 24 h, proteomic changes were partially restored with an exponential increase in HSP70 expression, and the cells restored their normal cellular morphology on the 9th day of recovery. Full proteomics data are available via ProteomeXchange with identifier PXD012159. The strategies of cellular defense and tolerance to both thermal conditions reflect the flexible adaptability of camel somatic cells to conserve life under extremely hot conditions.

Resveratrol inhibits adipocyte differentiation and cellular senescence of human bone marrow stromal stem cells.

Bone marrow adipose tissue (BMAT) is a unique adipose depot originating from bone marrow stromal stem cells (BMSCs) and regulates bone homeostasis and energy metabolism. An increased BMAT volume is observed in several conditions e.g. obesity, type 2 diabetes, osteoporosis and is known to be associated with bone fragility and increased risk for fracture. Therapeutic approaches to decrease the accumulation of BMAT are clinically relevant. In a screening experiment of natural compounds, we identified Resveratrol (RSV), a plant-derived antioxidant mediating biological effects via sirtuin- related mechanisms, to exert significant effects of BMAT formation. Thus, we examined in details the effects RSV on adipocytic and osteoblastic differentiation of tolermerized human BMSCs (hBMSC-TERT). RSV (1.0 µM) enhanced osteoblastic differentiation and inhibited adipocytic differentiation of hBMSC-TERT when compared with control and Sirtinol (Sirtuin inhibitor). Global gene expression profiling and western blot analysis revealed activation of a number of signaling pathways including focal adhesion kinase (FAK). Pharmacological inhibition of FAK using (PF-573228) and AKT inhibitor (LY-294002) (5µM), diminished RSV-induced osteoblast differentiation. In addition, RSV reduced the levels of senescence-associated secretory phenotype (SASP), gene markers associated with senescence (P53, P16, and P21), intracellular ROS levels and increased gene expression of enzymes protecting cells from oxidative damage (HMOX1 and SOD3). In vitro treatment of primary hBMSCs from aged patients characterized with high adipocytic and low osteoblastic differentiation ability with RSV, significantly enhanced osteoblast and decreased adipocyte formation when compared to hBMSCs from young donors. RSV targets hBMSCs and inhibits adipogenic differentiation and senescence-associated phenotype and thus a potential agent for treating conditions of increased BMAT formation.

MicroRNA-3148 acts as molecular switch promoting malignant transformation and adipocytic differentiation of immortalized human bone marrow stromal cells via direct targeting of the SMAD2/TGFß pathway.

MicroRNAs (miRs/miRNAs) play a key role in posttranscriptional regulation of gene expression and are implicated in a number of physiological and pathological conditions, including cellular malignant transformation. In the current study, we investigated the role of miR-3148 in regulating human stromal (mesenchymal) stem cell (hMSC) differentiation and transformation. Stable expression of miR-3148 in telomerized hMSC (hMSC-miR-3148) led to significant increase in in vitro adipocytic differentiation and suppression of osteoblastic differentiation. Concordantly, global gene expression profiling revealed significant enrichment in cholesterol biosynthesis pathway, and pathways related to enhanced cell movement and survival, whereas processes related to bone and connective tissue developments, cell death, apoptosis, and necrosis were downregulated. Global proteomic analysis using 2D-DIGE followed by mass spectrometry (MS) revealed significant changes in protein expression in hMSC-miR-3148 and enrichment in protein networks associated with carcinogenesis. Functional studies revealed that hMSC-miR-3148 exhibited enhanced in vitro cell proliferation, colony formation, migration, invasion, sphere formation, doxorubicin resistance, and increased active number of cells in S and G2/M cell cycle phases and formed sarcoma-like tumors with adipocyte infiltration when implanted into immunocompromised mice. SMAD2 was identified as bone fide gene target for miR-3148 using qRT-PCR, Western blotting, and UTR-based reporter assay. In agreement with our data, SMAD2 expression was downregulated in 47% of patients with soft tissue sarcoma. Bioinformatics analysis revealed that elevated miR-3148 expression correlates with poor prognosis in several human cancer types, including sarcoma. Our study identified miR-3148 as factor regulating hMSC differentiation and is involved in promoting malignant transformation of telomerized hMSC.

CXCR7 signaling promotes breast cancer survival in response to mesenchymal stromal stem cell-derived factors.

The interaction between cancer cells and molecular cues provided by tumor stromal cells plays a crucial role in cancer growth and progression. We have recently reported that the outcome of interaction between tumor cells and stromal cells is dependent on the gene expression signature of tumor cells. In the current study, we observed that several cancer cell lines, e.g., MCF7 breast cancer line, exhibited growth advantage when cultured in the presence of conditioned media (CM) derived from human bone marrow stromal stem cells (hBMSCs). Regarding the underlying molecular mechanism, we have identified CXCR7 as highly expressed by MCF7 cells and that it mediated the enhanced growth in response to hBMSC CM. Regarding the clinical relevance, we found an inverse correlation between the level of tumor gene expression of CXCR7 in bladder, breast, cervical, kidney, liver, lung, pancreatic, stomach, and uterine cancers, and patients' overall survival. Interestingly, significant positive correlation between CXCR7 and CXCL12 gene expression (Pearson = 0.3, p = 2.0 × 10-16) was observed in breast cancer patients, suggesting a biological role for the CXCR7/CXCL12 genetic circuit in breast cancer biology. Our data provide insight into the molecular mechanisms by which stromal-derived microenvironmental cues mediate CXCR7 signaling and growth enhancement of breast cancer cells. Therapeutic targeting of this circuit might provide novel therapeutic opportunity for breast cancer.

Convergence of TGFß and BMP signaling in regulating human bone marrow stromal cell differentiation.

Targeting regulatory signaling pathways that control human bone marrow stromal (skeletal or mesenchymal) stem cell (hBMSC) differentiation and lineage fate determination is gaining momentum in the regenerative medicine field. Therefore, to identify the central regulatory mechanism of osteoblast differentiation of hBMSCs, the molecular phenotypes of two clonal hBMSC lines exhibiting opposite in vivo phenotypes, namely, bone forming (hBMSC+bone) and non-bone forming (hBMSC-Bone) cells, were studied. Global transcriptome analysis revealed significant downregulation of several TGFß responsive genes, namely, TAGLN, TMP1, ACTA2, TGFß2, SMAD6, SMAD9, BMP2, and BMP4 in hBMSC-Bone cells and upregulation on SERPINB2 and NOG. Transcriptomic data was associated with marked reduction in SMAD2 protein phosphorylation, which thereby implies the inactivation of TGFß and BMP signaling in those cells. Concordantly, activation of TGFß signaling in hBMSC-Bone cells using either recombinant TGFß1 protein or knockdown of SERPINB2 TGFß-responsive gene partially restored their osteoblastic differentiation potential. Similarly, the activation of BMP signaling using exogenous BMP4 or via siRNA-mediated knockdown of NOG partially restored the differentiation phenotype of hBMSC-Bone cells. Concordantly, recombinant NOG impaired ex vivo osteoblastic differentiation of hBMSC+Bone cells, which was associated with SERBINB2 upregulation. Our data suggests the existence of reciprocal relationship between TGFB and BMP signaling that regulates hBMSC lineage commitment and differentiation, whilst provide a plausible strategy for generating osteoblastic committed cells from hBMSCs for clinical applications.

Author Correction: Convergence of TGFß and BMP signaling in regulating human bone marrow stromal cell differentiation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.