SOX9 Protein Levels Research Articles

E6-AP/UBE3A Protein Acts as a Ubiquitin Ligase toward SOX9 Protein

SOX9 is a transcription factor that acts as a key regulator at various stages of cartilage differentiation. There is ample evidence that intracellular SOX9 protein levels are tightly regulated both by sumoylation and by degradation through the ubiquitin-proteasome pathway. Using a proteomics approach, here we report the identification of a SOX9-binding protein, E6-AP/UBE3A, that may act as a ubiquitin ligase toward Sox9. E6-AP bound SOX9 through the region consisting mostly of its high mobility group domain in vitro. In nuclear lysates, FLAG-tagged E6-AP coprecipitated with Sox9 and its high mobility group domain. This finding was estimated using nuclear lysates from a chondrocytic cell line that endogenously expresses E6-AP and SOX9. Accordingly, ectopically expressed E6-AP and SOX9 colocalized in the nucleus. We show that E6-AP ubiquitinates SOX9 in vitro and in vivo and that SOX9 levels are enhanced after addition of the proteasome inhibitor bortezomib. Similar, siRNA knockdown of E6-AP and the E2 ligase Ubc9 increased cellular SOX9 amounts, supporting the notion that SOX9 may be ubiquitinated in hypertrophic chondrocytes by E6-AP and degraded by proteasomes. This is in accordance with the distribution of SOX9 levels, which are high in proliferating and prehypertrophic chondrocytes but low in hypertrophic chondrocytes, whereas E6-AP levels are high in hypertrophic chondrocytes and low in prehypertrophic chondrocytes. Furthermore, E6-AP-deficient mice showed SOX9 accumulation in chondrocytes and the brain. These findings support the concept that E6-AP regulates SOX9 levels in developing cartilage by acting as a ubiquitin ligase.

A study of embryonic stem cell‐related proteins in human astrocytomas: Identification of Nanog as a predictor of survival

Recent studies suggest that the regulatory networks controlling the functions of stem cells during development may be abnormally active in human cancers. An embryonic stem cell (ESC) gene signature was found to correlate with a more undifferentiated phenotype of several human cancer types including gliomas, and associated with poor prognosis in breast cancer. In the present study, we used tissue microarrays of 80 low-grade (WHO Grade II) and 98 high-grade human gliomas (WHO Grades III and IV) to investigate the presence of the ESC-related proteins Nanog, Klf4, Oct4, Sox2 and c-Myc by immunohistochemistry. While similar patterns of co-expressed proteins between low- and high-grade gliomas were present, we found up-regulated protein levels of Nanog, Klf4, Oct4 and Sox2 in high-grade gliomas. Survival analysis by Kaplan-Meier analysis revealed a significant shorter survival in the subgroups of low-grade astrocytomas (n = 42) with high levels of Nanog protein (p = 0.0067) and of Klf4 protein (p = 0.0368), in high-grade astrocytomas (n = 85) with high levels of Nanog (p = 0.0042), Klf4 (p = 0.0447), and c-Myc (p = 0.0078) and in glioblastomas only (n = 71) with high levels of Nanog (p = 0.0422) and of c-Myc (p = 0.0256). In the multivariate model, Nanog was identified as an independent prognostic factor in the subgroups of low-grade astrocytomas (p = 0.0039), high-grade astrocytomas (p = 0.0124) and glioblastomas only (p = 0.0544), together with established clinical variables in these tumors. These findings provide further evidence for the joint regulatory pathways of ESC-related proteins in gliomas and identify Nanog as one of the key players in determining clinical outcome of human astrocytomas.

Myosin VI Reduces Proliferation, but Not Differentiation, in Pluripotent P19 Cells

BackgroundWe have previously shown marked upregulation of the mRNA and corresponding protein for the cellular motor molecule myosin VI (Myo6) after an extremely traumatic stress experience, along with a delayed decrease in 5-bromo-2′-deoxyuridine incorporation in the murine hippocampus, a brain structure believed to undergo adult neurogenesis. In this study, we investigated the role of Myo6 in both proliferation and differentiation in pluripotent P19 cells by using stable transfection and RNA interference techniques.Methodology/Principal FindingsStable overexpression of Myo6 not only led to significant inhibition of the reducing activity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and the size of clustered aggregates in P19 cells, but also resulted in selectively decreased mRNA expression of the repressor type proneural gene Hes5 without affecting the expression of neuronal and astroglial marker proteins. In P19 cells transfected with Myo6 siRNA, by contrast, a significant increase was found in the size of aggregate and MTT reduction along with increased Sox2 protein levels, in addition to marked depletion of the endogenous Myo6 protein. In C6 glioma cells, however, introduction of Myo6 siRNA induced a drastic decrease in endogenous Myo6 protein levels without significantly affecting MTT reduction. The Ca2+ ionophore A23187 drastically increased the luciferase activity in P19 cells transfected with a Myo6 promoter reporter plasmid, but not in HEK293, Neuro2A and C6 glioma cells transfected with the same reporter.Conclusions/SignificanceThese results suggest that Myo6 may play a predominant pivotal role in the mechanism underlying proliferation without affecting differentiation to progeny lineages in pluripotent P19 cells.

Experimentally created unilateral anterior crossbite induces a degenerative ossification phenotype in mandibular condyle of growing Sprague‐Dawley rats

The effect of unilateral anterior crossbite on the remodelling of mandibular condyle needs to be investigated. This study aimed to investigate the effects of experimentally created unilateral anterior crossbite on the remodelling of mandibular condyle and explore the changes in the expression of relevant transcription factors and growth factors. The experimental unilateral anterior crossbite was created in 6-week-old female growing rats by bonding metal tubes to the left pairs of incisors. Remodelings of mandibular condylar cartilage was assessed histologically at 2, 4 and 8 weeks. Protein and mRNA levels of Sox9, runt-related transcription factor 2 (Runx2), Osterix (Osx), transforming growth factor beta 1 (TGFβ1), transforming growth factor beta receptor 2 (TGFβr2) and type X collagen (ColX) were investigated by immunohistochemistry and real-time PCR, while alkaline phosphatise (ALP) by histochemistry and real-time PCR. Decreased ratio of hypertrophic cartilage layer was noticed in the 4w experimental group versus controls. At all the time points, the expression of Sox9 and ALP increased but that of TGFβ1 and TGFβr2 decreased in experimental groups (P < 0·05). The expression of Runx2, Osx and Col X increased at 2w, but decrease at 4w (P < 0·05). The results that obvious cartilage degradation and altered expression of related transcription factors and growth factors were detected in the mandibular condyles of the experimental group suggested that the present unilateral anterior crossbite plays an adverse role in the TMJ, and thus leading to the degenerative endochondral ossification.

Molecular mechanism of indirubin-3′-monoxime and Matrine in the reversal of paclitaxel resistance in NCI-H520/TAX25 cell line

Background Multidrug resistance (MDR) is a main reason for paclitaxel (TAX) treatment failure. Indirubin-3′-monoxime (IRO) and Matrine are traditional Chinese medicines, which may reverse the resistance of tumor cells to some chemotherapy drugs, but the relationship between paclitaxel resistance and Matrine is still unclear. The aim of this study was to explore the potential molecular mechanism of IRO and Matrine in reversal of TAX resistance. Methods In this study, MTT assay was used to measure the non-cytotoxic dosage of IRO and Matrine on NCI-H520/TAX25 cells and determine the reversal extent of TAX resistance under non-toxic doses. In addition, RT-PCR and Western blotting were used to evaluate the mRNA expression and the protein level of survivin, Oct-4, and Sox-2 in NCI-H520/TAX25 cells using semi-quantitative methods. Results There was no obvious inhibition on sensitive cell strains and drug-resistant strains, when the final concentration was at lest 4 μmol/L for IRO and 100 μmol/L for Matrine. So 4 μmol/L of IRO and 100 μmol/L of Matrine were considered as the reversal dosage. When 4 μmol/L of IRO or 100 μmol/L of Matrine were used together with TAX, the sensitivity to TAX increased evidently in NCI-H520/TAX2 cells; the reversal rate of IRO and Matrine was about 1.92 (43.56/22.6 nmol/L) and 1.74 (43.56/25.0 nmol/L), respectively. The mRNA expression and the protein level of survivin, Oct-4, and Sox-2 in NCI-H520/TAX25 decreased significantly (P &lt;0.05) after addition of IRO or Matrine in TAX treatment, compared to that of TAX treatment alone. Conclusion The decrease in both mRNA expression and protein level of survivin, Oct-4, and Sox-2 might be the molecular mechanism, by which IRO and Matrine mediate the reversal of TAX resistance.

Sox9 Modulates Proliferation and Expression of Osteogenic Markers of Adipose-Derived Stem Cells (ASC)

Background: Mesenchymal stem cells (MSC) are promising tools for tissue-engineering and musculoskeletal regeneration. They reside within various tissues, like adipose tissue, periosteum, synovia, muscle, dermis, blood and bone marrow, latter being the most common tissue used for MSC isolation. A promising alternative source for MSC is adipose tissue due to better availability and higher yield of MSC in comparison to bone marrow. A drawback is the yet fragmentary knowledge of adipose-derived stem cell (ASC) physiology in order to make them a safe tool for in vivo application. Methods/Results: Here, we identified Sox9 as a highly expressed and crucial transcription factor in undifferentiated rat ASC (rASC). In comparison to rat bone marrow-derived stem cells (rBMSC), mRNA and protein levels of Sox9 were significantly higher in rASC. To study the role of Sox9 in detail, we silenced Sox9 with shRNA in rASC and examined proliferation, apoptosis and the expression of osteogenic differentiation markers. Our results clearly point to a difference in the expression profile of osteogenic marker genes between undifferentiated rASC and rBMSC in early passages. Sox9 silencing induced the expression of osteocalcin, Vegfα and Mmp13, and decreased rASC proliferation accompanied with an induction of p21 and cyclin D1 expression and delayed S-phase entry. Conclusions: We suggest a pro-proliferative role for Sox9 in undifferentiated rASC which may explain the higher proliferation rate of rASC compared to rBMSC. Moreover, we propose an osteogenic differentiation delaying role of Sox9 in rASC which suggests that Sox9 expression is needed to maintain rASC in an undifferentiated, proliferative state.

SOX2 expression is upregulated in adult spinal cord after contusion injury in both oligodendrocyte lineage and ependymal cells

The upregulation of genes normally associated with development may occur in the adult after spinal cord injury (SCI). To test this, we performed real-time RT-PCR array analysis of mouse spinal cord mRNAs comparing embryonic day (E)14.5 spinal cord with intact adult and adult cord 1 week after a clinically relevant standardized contusion SCI. We found significantly increased expression of a large number of neural development- and stem cell-associated genes after SCI. These included Sox2 (sex determining region Y-box 2), a transcription factor that regulates self-renewal and potency of embryonic neural stem cells and is one of only a few key factors needed to induce pluripotency. In adult spinal cord of Sox2-EGFP mice, Sox2-EGFP was found mainly in the ependymal cells of the central canal. After SCI, both mRNA and protein levels of Sox2 were significantly increased at and near the injury site. By 1 day, Sox2 was upregulated in NG2(+) oligodendrocyte progenitor cells (OPC) in the spared white matter. By 3 days, Sox2-EGFP ependymal cells had increased proliferation and begun to form multiple layers and clusters of cells in the central lesion zone of the cord. Expression of Sox2 by NG2(+) cells had declined by 1 week, but increased numbers of other Sox2-expressing cells persisted for at least 4 weeks after SCI in both mouse and rat models. Thus, SCI upregulates many genes associated with development and neural stem cells, including the key transcription factor Sox2, which is expressed in a pool of cells that persists for weeks after SCI.

Fibroblast Growth Factor Receptor 2 Homodimerization Rapidly Reduces Transcription of the Pluripotency Gene Nanog without Dissociation of Activating Transcription Factors

Nanog or Gata6-positive cells co-exist and are convertible within the inner cell mass of murine blastocysts and embryonic stem (ES) cells. Previous studies demonstrate fibroblast growth factor receptor 2 (FGFR2) triggers Nanog gene down-regulation and differentiation to primitive endoderm (PE); however, the underlying mechanisms responsible for reversible and fluctuating cell fate are poorly understood. Using an inducible FGFR2 dimerization system in ES cells, we demonstrate that FGFR2 activation rapidly down-regulated Nanog gene transcription through activation of the Mek pathway and subsequently differentiated ES cells into PE cells. FGFR2 rather selectively repressed the Nanog gene with minimal effect on other pluripotency genes, including Oct4 and Sox2. We determined the Nanog promoter region containing minimum Oct4/Sox2 binding sites was sufficient for this transcriptional down-regulation by FGFR2, when the reporter transgenes were integrated with insulators. Of interest, FGFR2-mediated Nanog transcriptional reduction occurred without dissociation of RNA polymerase II, p300, Oct4, Sox2, and Tet1 from the Nanog proximal promoter region and with no increase in repressive histone methylation marks or DNA methylation, implying the gene repression is in the early and transient phase. Furthermore, addition of a specific FGFR inhibitor readily reversed this Nanog repression status. These findings illustrate well how FGFR2 induces rapid but reversible Nanog repression within ES cells.

Abstract 427: Sox2 modulation of cancer stem cell behavior in GBM

Abstract Background and Objective: Glioblastoma (GBM) is the most aggressive primary brain tumor. GBM cells are phenotypically plastic and can present cancer stem cell (CSC) properties, such as the ability to self-renew and differentiate into the cells comprising the bulk of the tumor, phenocopying the parental tumor in mouse xenografts. GBM CSCs express neural stem cell (NSC) markers, including Sox2, a HMG-box transcription factor that regulates the expression of genes associated with self-renewal and differentiation. Culturing dissociated GBM tumors in serum-containing medium typically results in loss of the CSC phenotype and downregulation of Sox2, while serum-free neurosphere medium (NM) is used for selection and propagation of CSCs. We have previously identified a GBM specimen (GS1) which, contrary to the norm, retained Sox2 expression in low passage serum culture, along with the ability to dedifferentiate into neurospheres in vitro, and to form tumors in rodents (deCarvalho et al., Stem Cells 2010, 28:181-190). Here we investigate the endogenous expression of Sox2 in GBM biopsies, xenograft tumors, and cultured cells, and test the hypothesis that Sox2 may be a driver of the cancer stem cell phenotype in GBMs. Experimental Approaches: Endogenous expression of Sox2, downstream targets, and cell lineage markers, was determined by immunohistochemistry. miRNAs targeting Sox2 were stably expressed in GBM cells. The effect of Sox2 knockdown on clonogenicity, global gene expression, proliferation in vitro, tumor formation and morphology was determined. Results: Sox2 expression was heterogeneous within and among the 26 GBM surgical specimens tested, ranging from 1% to over 80% positive nuclei. Robust Sox2 expression was observed in over 80% of 56 GBM orthotopic xenografts analyzed. Sox2 positive cells co-expressed mesenchymal (vimentin), neural (nestin), and astrocytic (GFAP) markers. Knocking down Sox2 expression significantly impaired the ability of GS1 cells to form neurospheres, without affecting cell proliferation, significantly altered the expression of known target genes, such as Nestin, and genes in various signaling pathways, and affected xenograft tumor formation. Sox2 protein levels in GBMs were further regulated by post-transcriptional mechanisms. Conclusions: Sox2 is part of the signature gene set for the proneural GBM subclass. Our results show that Sox2 protein expression is ubiquitous in GBM tumors and xenografts, independent of patient survival or treatment status. Sox2 is a master regulator of embryonic and neural stem cell phenotype, and our data contributes to the view that it may have an important role in the plasticity of GBM cells in acquiring CSC phenotype, with possible implications for the pathology of GBMs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 427. doi:1538-7445.AM2012-427

Fibroblast Growth Factor-2 Primes Human Mesenchymal Stem Cells for Enhanced Chondrogenesis

Human mesenchymal stem cells (hMSCs) are multipotent cells capable of differentiating into a variety of mature cell types, including osteoblasts, adipocytes and chondrocytes. It has previously been shown that, when expanded in medium supplemented with fibroblast growth factor-2 (FGF-2), hMSCs show enhanced chondrogenesis (CG). Previous work concluded that the enhancement of CG could be attributed to the selection of a cell subpopulation with inherent chondrogenic potential. In this study, we show that FGF-2 pretreatment actually primed hMSCs to undergo enhanced CG by increasing basal Sox9 protein levels. Our results show that Sox9 protein levels were elevated within 30 minutes of exposure to FGF-2 and progressively increased with longer exposures. Further, we show using flow cytometry that FGF-2 increased Sox9 protein levels per cell in proliferating and non-proliferating hMSCs, strongly suggesting that FGF-2 primes hMSCs for subsequent CG by regulating Sox9. Indeed, when hMSCs were exposed to FGF-2 for 2 hours and subsequently differentiated into the chondrogenic lineage using pellet culture, phosphorylated-Sox9 (pSox9) protein levels became elevated and ultimately resulted in an enhancement of CG. However, small interfering RNA (siRNA)-mediated knockdown of Sox9 during hMSC expansion was unable to negate the prochondrogenic effects of FGF-2, suggesting that the FGF-2-mediated enhancement of hMSC CG is only partly regulated through Sox9. Our findings provide new insights into the mechanism by which FGF-2 regulates predifferentiation hMSCs to undergo enhanced CG.

MicroRNA-145 Regulates Chondrogenic Differentiation of Mesenchymal Stem Cells by Targeting Sox9

Chondrogenic differentiation of mesenchymal stem cells (MSCs) is accurately regulated by essential transcription factors and signaling cascades. However, the precise mechanisms involved in this process still remain to be defined. MicroRNAs (miRNAs) regulate various biological processes by binding target mRNA to attenuate protein synthesis. To investigate the mechanisms for miRNAs-mediated regulation of chondrogenic differentiation, we identified that miR-145 was decreased during transforming growth factor beta 3 (TGF-β3)-induced chondrogenic differentiation of murine MSCs. Subsequently, dual-luciferase reporter gene assay data demonstrated that miR-145 targets a putative binding site in the 3′-UTR of SRY-related high mobility group-Box gene 9 (Sox9) gene, the key transcription factor for chondrogenesis. In addition, over-expression of miR-145 decreased expression of Sox9 only at protein levels and miR-145 inhibition significantly elevated Sox9 protein levels. Furthermore, over-expression of miR-145 decreased mRNA levels for three chondrogenic marker genes, type II collagen (Col2a1), aggrecan (Agc1), cartilage oligomeric matrix protein (COMP), type IX collagen (Col9a2) and type XI collagen (Col11a1) in C3H10T1/2 cells induced by TGF-β3, whereas anti-miR-145 inhibitor increased the expression of these chondrogenic marker genes. Thus, our studies demonstrated that miR-145 is a key negative regulator of chondrogenic differentiation by directly targeting Sox9 at early stage of chondrogenic differentiation.

Pluripotency Factors in Embryonic Stem Cells Regulate Differentiation into Germ Layers

Cell fate decisions are fundamental for development, but we do not know how transcriptional networks reorganize during the transition from a pluripotent to a differentiated cell state. Here, we asked how mouse embryonic stem cells (ESCs) leave the pluripotent state and choose between germ layer fates. By analyzing the dynamics of the transcriptional circuit that maintains pluripotency, we found that Oct4 and Sox2, proteins that maintain ESC identity, also orchestrate germ layer fate selection. Oct4 suppresses neural ectodermal differentiation and promotes mesendodermal differentiation; Sox2 inhibits mesendodermal differentiation and promotes neural ectodermal differentiation. Differentiation signals continuously and asymmetrically modulate Oct4 and Sox2 protein levels, altering their binding pattern in the genome, and leading to cell fate choice. The same factors that maintain pluripotency thus also integrate external signals and control lineage selection. Our study provides a framework for understanding how complex transcription factor networks control cell fate decisions in progenitor cells.

An EGFR-ERK-SOX9 Signaling Cascade Links Urothelial Development and Regeneration to Cancer

Like many carcinomas, urothelial carcinoma (UroCa) is associated with chronic injury. A better understanding of this association could inform improved strategies for preventing and treating this disease. We investigated the expression, regulation, and function of the transcriptional regulator SRY-related high-mobility group box 9 (Sox9) in urothelial development, injury repair, and cancer. In mouse bladders, Sox9 levels were high during periods of prenatal urothelial development and diminished with maturation after birth. In adult urothelial cells, Sox9 was quiescent but was rapidly induced by a variety of injuries, including exposure to the carcinogen cyclophosphamide, culture with hydrogen peroxide, and osmotic stress. Activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was required for Sox9 induction in urothelial injury and resulted from activation of the epidermal growth factor receptor (Egfr) by several Egfr ligands that were dramatically induced by injury. In UroCa cell lines, SOX9 expression was constitutively upregulated and could be suppressed by EGFR or ERK1/2 blockade. Gene knockdown showed a role for SOX9 in cell migration and invasion. Accordingly, SOX9 protein levels were preferentially induced in invasive human UroCa tissue samples (n = 84) compared with noninvasive cancers (n = 56) or benign adjacent urothelium (n = 49). These results identify a novel, potentially oncogenic signaling axis linking urothelial injury to UroCa. Inhibiting this axis is feasible through a variety of pharmacologic approaches and may have clinical utility.

Abstract 4381: EGFR-Src signaling regulates self-renewal of cancer stem like cells from NSCLC through Sox2

Abstract NSCLC is the most common form of lung cancer with 5-year survival rate of approximately 10%. Cancer stem cell model has been proposed for tumor-hetrogeniety, carcinogenesis and relapse after therapy. According to this model, cancer stem like cells (CSCs) are defined as self-renewing tumor cells, able to initiate and maintain the tumor. Here, we isolated CSCs based on “side populaton” (SP) phenotype, a functional property of adult stem cells to efflux Hoechst-33352 dye through ABCG2 transporter. Irrespective of genotype of the cells, we observed SP-cells in H358, H292, H1650, H1975, A549 and H460 cell lines. SP phenotype was also displayed by clinical-human xenografts, which could be specifically blocked by ABCG2 inhibitor. Relative tumorigenic potential of SP and MP (main population) cells was determined by subcutaneous or orthotopic xenograft-implantation in SCID mice. As low as 1×103 SP-cells isolated from H1650 cells could form subcutaneous tumor. Similarly, 5×104 SP-cells from A549 could establish orthotopic tumors in lung. Flow-cytometry analysis of subcutaneous tumors demonstrated the asymmetric division of SP-cells generating MP-cells within the tumors. In vitro analysis showed higher expression of ABCG2 and anti-apoptotic protein MCL1 in SP-cells as compared to MP cells. Further, SP-cells displayed higher expression of mesenchymal marker vimentin and lower levels of epithelial marker E-cadherin, suggesting the EMT-like features in SP-cells. SP-cells were found to express embryonic self-renewal factors Oct4, Sox2 and Nanog and demonstrated self-renewal capability by growing as suspended spheres in serum free, stem cell-selective medium. To successfully eliminate these CSCs from tumors, it is necessary to understand the mechanisms facilitating its self-renewal. Based on the favorable clinical outcome of EGFR- and Src-targeted therapy against certain NSCLCs, we hypothesized that the activated EGFR and Src signaling might play important roles in CSCs of NSCLC. This hypothesis was tested using biochemical as well as genetic inhibitor tools against EGFR and Src. SiRNA against EGFR significantly blocked the SP phenotype by downregulating ABCG2 protein levels in all the tested cell lines. Importantly, inhibitor of EGFR (Gefitinib, Erlotinib, BIBW2992) and Src (Dasatinib, PP2) completely blocked the self-renewal capability of SP-cells, as assessed by sphere formation assays. Further, blocking of EGFR and Src signaling by SiRNA or inhibitors resulted in downregulation of the protein levels of Sox2 in SP cells. Importantly, SiRNA against Sox2 significantly blocked the SP phenotype as well as self-renewal capacity of SP-cells. Our findings reveal an important role of EGFR-Src signaling axis in self-renewal and expansion of NSCLC-CSCs by regulating the Sox2 activity in the cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4381. doi:10.1158/1538-7445.AM2011-4381

MicroRNA-126 Inhibits SOX2 Expression and Contributes to Gastric Carcinogenesis

BackgroundSRY (sex-determining region Y)-box 2 (SOX2) is a crucial transcription factor for the maintenance of embryonic stem cell pluripotency and the determination of cell fate. Previously, we demonstrated that SOX2 plays important roles in growth inhibition through cell cycle arrest and apoptosis, and that SOX2 expression is frequently down-regulated in gastric cancers. However, the mechanisms underlying loss of SOX2 expression and its target genes involved in gastric carcinogenesis remain largely unknown. Here, we assessed whether microRNAs (miRNAs) regulate SOX2 expression in gastric cancers. Furthermore, we attempted to find downstream target genes of SOX2 contributing to gastric carcinogenesis.Methodology/Principal FindingsWe performed in silico analysis and focused on miRNA-126 (miR-126) as a potential SOX2 regulator. Gain- and loss-of function experiments and luciferase assays revealed that miR-126 inhibited SOX2 expression by targeting two binding sites in the 3′-untranslated region (3′-UTR) of SOX2 mRNA in multiple cell lines. In addition, miR-126 was highly expressed in some cultured and primary gastric cancer cells with low SOX2 protein levels. Furthermore, exogenous miR-126 over-expression as well as siRNA-mediated knockdown of SOX2 significantly enhanced the anchorage-dependent and -independent growth of gastric cancer cell lines. We next performed microarray analysis after SOX2 over-expression in a gastric cancer cell line, and found that expression of the placenta-specific 1 (PLAC1) gene was significantly down-regulated by SOX2 over-expression. siRNA- and miR-126-mediated SOX2 knockdown experiments revealed that miR-126 positively regulated PLAC1 expression through suppression of SOX2 expression in gastric cancer cells.ConclusionsTaken together, our results indicate that miR-126 is a novel miRNA that targets SOX2, and PLAC1 may be a novel downstream target gene of SOX2 in gastric cancer cells. These findings suggest that aberrant over-expression of miR-126 and consequent SOX2 down-regulation may contribute to gastric carcinogenesis.

Akt Increases Sox2 Expression in Adult Hippocampal Neural Progenitor Cells, but Increased Sox2 Does Not Promote Proliferation

Multiple extracellular factors have been shown to modulate adult hippocampal neural progenitor cell (NPC) proliferation and self-renewal, and we have previously shown that Akt is an important mediator of the effects of these extracellular factors on NPC proliferation and differentiation. However, very little work has investigated how and whether Akt is involved in maintaining the multipotency of these cells. Here we demonstrate that Akt promotes expression of Sox2, a core transcription factor important for the self-renewal of NPCs. Retroviral-mediated overexpression of wild-type Akt increased Sox2 protein expression, particularly under conditions that promote cell differentiation, whereas Akt inhibition decreased Sox2. Similarly, quantitative reverse transcription (RT)-PCR in differentiating cultures indicated that Akt rescued Sox2 mRNA to levels present under conditions that promote cell proliferation. Additionally, pharmacological inhibition of Akt did not affect Sox2 protein levels in cells constitutively expressing Sox2 from a retroviral vector, indicating that Akt does not affect Sox2 protein stability. Further, in contrast to Akt overexpression, Sox2 overexpression does not increase NPC viable cell number or proliferation yet does inhibit differentiation. Collectively, these results indicate that Akt promotes cell proliferation and maintenance of a multipotent state via two downstream paths.

Comparative Characterization of Porcine Mesenchymal Stem Cells Derived from Bone Marrow Extract and Skin Tissues

Mesenchymal stem cells (MSCs) offer a great promise for regenerative medicine. Present study compared the characterization of porcine MSCs (pMSCs) derived from bone marrow extract with adult ear and fetal skin-derived cells on morphology, cell growth, alkaline phosphatase activity, proliferation ability, expression of cluster of differentiation (CD) markers (CD29, 45, and 90), cell cycle, protein and mRNA levels of Oct-4, Sox-2, and Nanog, and lineage differentiation ability. Skin-derived cells exhibited alkaline phosphatase activity and differentiation ability like pMSCs. pMSCs possessed a longer doubling time than skin-derived cells, and there was no difference in the ratio of G0/G1 phase between pMSCs and skin-derived cells. Except for CD29 and 90, all cells were found negative for CD45. Protein and mRNA expression of Oct-4, Sox-2 and Nanog were observed with similar intensity in all cells. Taken together, pMSCs and skin-derived cells revealed similar characteristics, and suggested the possible supportive role of skin-derived cells with MSCs for the regeneration of damaged tissues in cell-based therapies.

Sox2 Is Essential for Formation of Trophectoderm in the Preimplantation Embryo

BackgroundIn preimplantation mammalian development the transcription factor Sox2 (SRY-related HMG-box gene 2) forms a complex with Oct4 and functions in maintenance of self-renewal of the pluripotent inner cell mass (ICM). Previously it was shown that Sox2−/− embryos die soon after implantation. However, maternal Sox2 transcripts may mask an earlier phenotype. We investigated whether Sox2 is involved in controlling cell fate decisions at an earlier stage.Methods and FindingsWe addressed the question of an earlier role for Sox2 using RNAi, which removes both maternal and embryonic Sox2 mRNA present during the preimplantation period. By depleting both maternal and embryonic Sox2 mRNA at the 2-cell stage and monitoring embryo development in vitro we show that, in the absence of Sox2, embryos arrest at the morula stage and fail to form trophectoderm (TE) or cavitate. Following knock-down of Sox2 via three different short interfering RNA (siRNA) constructs in 2-cell stage mouse embryos, we have shown that the majority of embryos (76%) arrest at the morula stage or slightly earlier and only 18.7–21% form blastocysts compared to 76.2–83% in control groups. In Sox2 siRNA-treated embryos expression of pluripotency associated markers Oct4 and Nanog remained unaffected, whereas TE associated markers Tead4, Yap, Cdx2, Eomes, Fgfr2, as well as Fgf4, were downregulated in the absence of Sox2. Apoptosis was also increased in Sox2 knock-down embryos. Rescue experiments using cell-permeant Sox2 protein resulted in increased blastocyst formation from 18.7% to 62.6% and restoration of Sox2, Oct4, Cdx2 and Yap protein levels in the rescued Sox2-siRNA blastocysts.Conclusion and SignificanceWe conclude that the first essential function of Sox2 in the preimplantation mouse embryo is to facilitate establishment of the trophectoderm lineage. Our findings provide a novel insight into the first differentiation event within the preimplantation embryo, namely the segregation of the ICM and TE lineages.

PKCdelta is a positive regulator of chondrogenesis in chicken high density micromass cell cultures

We aimed to elucidate the role of the Ca-independent PKC isoenzyme PKCdelta in the regulation of spontaneous in vitro chondrogenesis occurring in a 6-day-long culturing period in chicken limb bud-derived high density cell cultures (HDC). PKCdelta expression and activity were detectable throughout the entire culturing period with a peak on days 2 and 3, when most of the chondroblasts differentiate. To inhibit the activity of PKCdelta, either the natural compound rottlerin was transiently applied to the culture medium of HDC in 2.5, 5 or 10 μM concentrations, or gene silencing was performed by using PKCdelta shRNA. Rottlerin significantly reduced the overall PKC activity in enzyme activity assays of cell-free samples of untreated control HDC, probably via the inhibition of PKCdelta. On the contrary, we were unable to detect any consistent change of PKC enzyme activity assayed in samples of HDC treated with rottlerin during culturing. PKCdelta gene silencing resulted in a significantly lower PKC activity. Both rottlerin and PKCdelta shRNA caused a severe reduction in cartilage formation, furthermore protein and phospho-protein levels of Sox9, the key transcription factor of chondrogenesis, were also significantly decreased. Rottlerin lowered, while PKCdelta gene silencing elevated the phosphorylation status of ERK1/2. Our data suggest that PKCdelta stimulates chondrogenesis via influencing Sox9 and ERK1/2 phosphorylation, but the inhibition of cartilage formation in the rottlerin-treated HDC is probably PKCdelta independent and rottlerin might have different effects when applied to cells or to an in vitro enzyme activity assay.

Abstract 4029: Sox9 functions downstream of Hedgehog-Gli1 in epithelial transformation

Abstract The Sonic Hedgehog (Shh) signaling pathway plays critical roles in cell fate specification during embryonic and fetal development and in adult tissues. The extracellular morphogen, Shh, signals through Gli family zinc finger transcription factors to regulate the expression of downstream target genes. Constitutive Shh-Gli signaling is implicated in the pathogenesis of several human cancers, including cutaneous basal cell carcinoma (BCC), prostate cancer and medulloblastoma. How Gli can signal through downstream genes to induce malignant transformation remains poorly understood. In this work, we performed microarray analysis to reveal the gene expression profile after tetracycline-regulated induction of Gli1 for 3 or 6 hours in cultured epithelial cells, identifying multiple known Shh target genes. Among the most rapidly induced genes was Sox9, a member of SRY family of transcription factors. Sox9 is essential for male sex determination and is a master regulator of chondrogenesis. Sox9 is also well established as a Shh-Gli target gene, but how it functions in Shh-Gli signaling is not well understood. We found that Sox9 mRNA and protein levels are greatly increased after short-term or long-term Gli1 expression in a rat culture model of epithelial cells, termed RK3E, and in mouse skin. As shown by reporter assays, Sox9 transcriptional activity was increased by approximately 18-fold in RK3E cells transformed by Gli1. To enable knockdown studies in rat cells, we defined the full-length rat Sox9 coding sequence. Subsequently, suppression of rat Sox9 by siRNA indicated an essential role for this Gli-regulated gene in malignant transformation of RK3E in vitro. Specifically, shRNA plasmids that suppress Sox9 could dramatically reduced the transforming activity of Gli1. In contrast, there was little effect on other transforming oncoproteins including H-Ras and ErbB2. These results establish Sox9 induction as an early response to Gli1, and demonstrate that induction of Sox9 is essential for transformation by Gli1. Further characterization of the Sox9 knockdown phenotype is in progress and will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4029.