Mesenchymal Cell State Research Articles

BRD4 mediates NF-κB-dependent epithelial-mesenchymal transition and pulmonary fibrosis via transcriptional elongation.

Chronic epithelial injury triggers a TGF-β-mediated cellular transition from normal epithelium into a mesenchymal-like state that produces subepithelial fibrosis and airway remodeling. Here we examined how TGF-β induces the mesenchymal cell state and determined its mechanism. We observed that TGF-β stimulation activates an inflammatory gene program controlled by the NF-κB/RelA signaling pathway. In the mesenchymal state, NF-κB-dependent immediate-early genes accumulate euchromatin marks and processive RNA polymerase. This program of immediate-early genes is activated by enhanced expression, nuclear translocation, and activating phosphorylation of the NF-κB/RelA transcription factor on Ser276, mediated by a paracrine signal. Phospho-Ser276 RelA binds to the BRD4/CDK9 transcriptional elongation complex, activating the paused RNA Pol II by phosphorylation on Ser2 in its carboxy-terminal domain. RelA-initiated transcriptional elongation is required for expression of the core epithelial-mesenchymal transition transcriptional regulators SNAI1, TWIST1, and ZEB1 and mesenchymal genes. Finally, we observed that pharmacological inhibition of BRD4 can attenuate experimental lung fibrosis induced by repetitive TGF-β challenge in a mouse model. These data provide a detailed mechanism for how activated NF-κB and BRD4 control epithelial-mesenchymal transition initiation and transcriptional elongation in model airway epithelial cells in vitro and in a murine pulmonary fibrosis model in vivo. Our data validate BRD4 as an in vivo target for the treatment of pulmonary fibrosis associated with inflammation-coupled remodeling in chronic lung diseases.

Transcriptional targets of TWIST1 in the cranial mesoderm regulate cell-matrix interactions and mesenchyme maintenance

TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. We have previously shown that, in the absence of TWIST1, cells within the cranial mesoderm adopt an abnormal epithelial configuration via a process reminiscent of a mesenchymal to epithelial transition (MET). Here, we show by gene expression analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. ChIP-seq was used to identify TWIST1-binding sites in an in vitro model of a TWIST1-dependent mesenchymal cell state, and the data were combined with the transcriptome data to identify potential target genes. Three direct transcriptional targets of TWIST1 (Ddr2, Pcolce and Tgfbi) were validated by ChIP-PCR using mouse embryonic tissues and by luciferase assays. Our findings reveal that the mesenchymal properties of the cranial mesoderm are likely to be regulated by a network of TWIST1 targets that influences the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures.

Abstract IA10: Sox10 regulates stem/progenitor and mesenchymal cell states in mammary epithelial cells

Abstract To discover mechanisms that mediate the initiation and progression of aggressive and stem-like breast cancers, we characterized signaling networks that are present in the mammary stem cells responsible for fetal and adult mammary development. These analyses identified a signaling axis between FGF signaling and the transcription factor, Sox10. Here we report that Sox10 is specifically expressed in mammary cells that exhibit the highest levels of stem/progenitor activity. This includes fetal and adult mammary cells in vivo and mammary organoids in vitro. Sox10 is functionally relevant, as its deletion reduces stem/progenitor competence, while its overexpression increases stem/progenitor activity. Intriguingly, we also discover that Sox10 overexpression elicits epithelial-to-mesenchymal transition (EMT) in mammary organoids in an FGF signaling-dependent manner. Further, modulation of Sox10 levels can induce sequential EMT, migratory, and clonogenic behaviors that strikingly resemble proposed mechanisms of metastasis. Consistent with these findings, we find that Sox10 is preferentially expressed in the most stem- and EMT-like triple negative breast cancer subtypes, and that Sox10+ tumor cells utilize FGF signaling for growth and invasive behaviors, which suggest that Sox10 may reprise these same functions during tumorigenesis. Collectively, these results demonstrate a signaling mechanism through which stem and mesenchymal-like states are acquired in mammary cells, and indicate possible therapeutic targets to counter these functions in breast cancers for which targeted therapies are currently unavailable. Citation Format: Christopher Dravis, Geoffrey M. Wahl. Sox10 regulates stem/progenitor and mesenchymal cell states in mammary epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr IA10.

Interplay Between Transcription Factors and MicroRNAs Regulating Epithelial-Mesenchymal Transitions in Colorectal Cancer.

The epithelial-mesenchymal-transition (EMT) represents a morphogenetic program involved in developmental processes such as gastrulation and neural crest formation. The EMT program is co-opted by epithelial tumor cells and endows them with features necessary for spreading to distant sites, such as invasion, migration, apoptosis resistance and stemness. Thereby, EMT facilitates metastasis formation and therapy resistance. A growing number of transcription factors has been implicated in the regulation of EMT. These include EMT-inducing transcription factors (EMT-TFs), the most prominent being SNAIL, SLUG, ZEB1, ZEB2 and TWIST, and negative regulators of EMT, such as p53. Furthermore, a growing number of microRNAs, such as members of the miR-200 and miR-34 family, have been characterized as negative regulators of EMT. EMT-TFs and microRNAs, such as ZEB1/2 and miR-200 or SNAIL and miR-34, are often engaged in double-negative feedback loops forming bistable switches controlling the transitions from epithelial to the mesenchymal cell states. Within this chapter, we will provide a comprehensive overview over the transcription factors and microRNAs that have been implicated in the regulation of EMT in colorectal cancer. Furthermore, we will highlight the regulatory connections between EMT-TFs and miRNAs to illustrate common principles of their interaction that regulate EMTs.

Abstract A21: Proteomic profiling to elucidate intratumoral heterogeneity and cancer evolution in lung cancer

Abstract Tumors often display a high degree of intratumoral heterogeneity as manifested by dynamic changes in gene expression, protein expression, and on gross examination of histology, among many other features. Clinically, this underlying heterogeneity can drive tumor evolution and progression towards a more aggressive neoplastic state and a worse prognosis for patients; therefore, identifying the diverse composition of a tumor for early risk stratification is of critical importance. To elucidate intratumoral heterogeneity and intracellular hierarchy in a novel manner, we first conducted a low-cost quantitative proteomics analysis using MALDI-TOF mass spectrometry on over 1900 samples from different histological regions of individual tumors from 35 lung cancer patients, as well as from 3 mesenchymal stem cell samples. The histologies identified were acinar, basal cells, bronchial epithelium, lepidic, complex gland, micropapillary, near tumor normal, normal alveolar, papillary, papillary lepidic, papillary mucinous, and solid. Patient-specific information including survival status, sex, age, smoking status, SUV by FDG-PET scan, tumor size, EGFR, KRAS, and ERCC1 mutation status, among other variables was obtained. We then compared the proteomes derived from each tumor to the stem cell proteomes, and using computational strategies, mapped the distance of each histological sample from the mesenchymal stem cell state; using clustering techniques, we organized the major histological subtypes into a phylogenetic tree from stem cells to normal lung. We hypothesized that by applying and improving upon map of tumor evolution based on the distance of each individual histological sample from a stem cell state. Apart from liquid tumors, there have thus far been limited studies on the prognostic significance of different subclones in solid tumors, and therefore we treated each histological sample as a subclone within each patient. We also aimed to identify survival-associated subclones and prognostic molecular signatures across combinations of subclones. Identifying these subclones may provide insight into malignant micrometastases to other organs. Using co-expression network analysis, we further pinpointed distinctive significantly dysregulated co-regulatory protein networks within each histological subtype. Based on these networks, we sought to identify important hub proteins within each histology. Ultimately, using proteomic profiling in solid tumors can be a novel approach in functionally characterizing intratumoral heterogeneity, and may allow for a more robust analysis of the diverse molecular expression of single tumor samples. Our results may help inform the field of targeted broad-scale proteomics profiling for therapeutic use. Citation Format: Charlotte Lee, Hua-Jun Wu, Andre L. Moreira, Erin H. Seeley, Callee Walsh, Robert J. Downey, Franziska Michor. Proteomic profiling to elucidate intratumoral heterogeneity and cancer evolution in lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A21.

Abstract A1-19: A comprehensive transcriptional portrait of human cancer cell lines

Abstract Tumor-derived cell lines have served as vital models to advance our understanding of oncogene function and therapeutic responses. They represent a wide range of tumor types and show great diversity in their response to perturbations, thereby providing an important model system to capture the disease heterogeneity that defines the cancer patient population. Although substantial effort has been directed to defining the genomic constitution of cancer cell line panels, the transcriptome remains understudied. Most previous efforts have used probe-based hybridization technologies, which fail to reveal certain RNA features, such as oncogenic fusion transcripts, non-coding transcripts, non-human RNAs and expressed single nucleotide variants (SNVs). To shed light on these important features of human cancer cell lines we generated comprehensive sequence analysis of the poly-A+ transcriptome of 675 commonly used cancer cell lines with matched SNP array data. Analysis of the RNA-seq data revealed a comprehensive portrait of expression in these cell lines. Many cell lines, despite their unique names, share a common origin. We identified 109 cell lines in our study with > 85% concordance based on SNP array measurement. All subsequent analyses were performed using a reduced set of cell lines without genomically highly similar lines. Examining the expression of genes over the entire set of cell lines showed a dominant epithelial/mesenchymal gene expression signature dividing solid tumor cell lines. Interestingly, we identified several long intergenic non-coding (linc)RNAs associated with either epithelial or mesenchymal cell state. Global correlation analysis led to the identification of new gene regulation patterns for the known oncogenes MET and EGFR. Detection of non-human sequences allows for the detection of pathogenic sequences present in cancer cell lines. We identified presence of sequence belonging to known oncogenic viruses – such as human papilloma virus and Epstein-Barr virus – as well as viruses that may be remnants of viral transfection experiments or indicative of persistent contamination. We used a splicing-aware mapping approach to 2,200 predicted in-frame gene fusion pairs. Of the 2,200 gene fusions catalogued, 1,435 consist of genes not previously found in fusions, providing many leads for further investigation. We combined the fusion results from the cancer cell lines with fusions detected in 6,730 primary tumors from The Cancer Genome Atlas (TCGA) and a database of previously described fusion genes found in literature. In total, we identified 232 cell lines that can serve as a model system to investigate fusion genes detected in clinical samples. Finally, we combine multiple genome and transcriptome features in a pathway-based approach to enhance prediction of response to targeted therapeutics. We combined multiple types of genomic aberration (mutation, copy number, fusion and overexpression) of given pathway genes to score every cell line as pathway aberrant or normal. Prediction of drug sensitivity for PIK3CA inhibitors, MEK inhibitors and an FGFR inhibitor was improved by an order of magnitude for the pathway-based approach compared to single gene predictors. It is becoming increasingly apparent that cell lines exhibit significant genomic and transcriptomic diversity comparable to the diversity observed among solid tumors. Comprehensive characterization of existing cell lines will allow selection of appropriate cell line models for biological studies and drug discovery. Our study greatly expands the current understanding of human cancer cell lines and provides a foundation for many additional discoveries that will enable further cancer studies and the successful development of novel therapeutics. Citation Format: Christiaan Klijn, Jeff Settleman, Somasekar Seshagiri, Zemin Zhang. A comprehensive transcriptional portrait of human cancer cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-19.

Sox10 Regulates Stem/Progenitor and Mesenchymal Cell States in Mammary Epithelial Cells.

To discover mechanisms that mediate plasticity in mammary cells, we characterized signaling networks that are present in the mammary stem cells responsible for fetal and adult mammary development. These analyses identified a signaling axis between FGF signaling and the transcription factor Sox10. Here, we show that Sox10 is specifically expressed in mammary cells exhibiting the highest levels of stem/progenitor activity. This includes fetal and adult mammary cells in vivo and mammary organoids in vitro. Sox10 is functionally relevant, as its deletion reduces stem/progenitor competence whereas its overexpression increases stem/progenitor activity. Intriguingly, we also show that Sox10 overexpression causes mammary cells to undergo a mesenchymal transition. Consistent with these findings, Sox10 is preferentially expressed in stem- and mesenchymal-like breast cancers. These results demonstrate a signaling mechanism through which stem and mesenchymal states are acquired in mammary cells and suggest therapeutic avenues in breast cancers for which targeted therapies are currently unavailable.

Microarray profiling to analyze the effect of Snai1 loss in mouse intestinal epithelium.

Epithelial stem cells from a variety of tissues have been shown to express genes linked to mesenchymal cell states. The Snail family of transcriptional factors has long been regarded as a marker of mesenchymal cells, however recent studies have indicated an involvement in regulation of epithelial stem cell populations. Snai1 is expressed in the stem cell population found at the base of the mouse small intestinal crypt that is responsible for generating all differentiated cell types of the intestinal epithelium. We utilized an inducible Cre recombinase approach in the intestinal epithelium combined with a conditional floxed Snai1 allele to induce knockout of gene function in the stem cell population. Loss of Snai1 resulted in loss of crypt base columnar cells and a failure to induce a proliferative response following radiation damage. We induced Snai1 loss in cultured organoids that had been derived from epithelial cells and compared gene expression to organoids with functional Snai1. Here we describe in detail the methods for generation of knockout organoids and analysis of microarray data that has been deposited in Gene Expression Omnibus (GEO):GSE65005.

Sox10 Regulates Stem/Progenitor and Mesenchymal Cell States in Mammary Epithelial Cells

Sox10 Regulates Stem/Progenitor and Mesenchymal Cell States in Mammary Epithelial Cells

Altered Transcriptional Control Networks with Trans-Differentiation of Isogenic Mutant-KRas NSCLC Models.

Background: The capacity of cancer cells to undergo epithelial mesenchymal trans-differentiation has been implicated as a factor driving metastasis, through the acquisition of enhanced migratory/invasive cell programs and the engagement of anti-apoptotic mechanisms promoting drug and radiation resistance. Our aim was to define molecular signaling changes associated with mesenchymal trans-differentiation in two KRas mutant NSCLC models. We focused on central transcription and epigenetic regulators predicted to be important for mesenchymal cell survival.Experimental design: We have modeled trans-differentiation and cancer stemness in inducible isogenic mutant-KRas H358 and A549 non-small cell lung cell backgrounds. As expected, our models show mesenchymal-like tumor cells acquire novel mechanisms of cellular signaling not apparent in their epithelial counterparts. We employed large-scale quantitative phosphoproteomic, proteomic, protein–protein interaction, RNA-Seq, and network function prediction approaches to dissect the molecular events associated with the establishment and maintenance of the mesenchymal state.Results: Gene-set enrichment and pathway prediction indicated BMI1, KDM5B, RUNX2, MYC/MAX, NFκB, LEF1, and HIF1 target networks were significantly enriched in the trans-differentiation of H358 and A549 NSCLC models. Physical overlaps between multiple networks implicate NR4A1 as an overlapping control between TCF and NFκB pathways. Enrichment correlations also indicated marked decrease in cell cycling, which occurred early in the EMT process. RNA abundance time course studies also indicated early expression of epigenetic and chromatin regulators within 8–24 h, including CITED4, RUNX3, CMBX1, and SIRT4.Conclusion: Multiple transcription and epigenetic pathways where altered between epithelial and mesenchymal tumor cell states, notably the polycomb repressive complex-1, HP1γ, and BAF/Swi-Snf. Network analysis suggests redundancy in the activation and inhibition of pathway regulators, notably factors controlling epithelial cell state. Through large-scale transcriptional and epigenetic cell reprograming, mesenchymal trans-differentiation can promote diversification of signaling networks potentially important in resistance to cancer therapies.

Abstract CN06-03: Cell plasticity promotes drug resistance and pathway redundancy.

Abstract Multiple signal transduction pathways can be concurrently active within a single cell, and extensive crosstalk can occur between RTKs. Additionally, tumor tissues can be comprised of a heterogeneous collection of cell states utilizing distinct RTKs for maintenance of tumor cell growth and survival. As a consequence of this complexity, many tumors may be only partially sensitive to single agent therapies and would require the interdiction of multiple RTKs and other protein signaling targets for optimal anticancer therapy. Understanding pathway crosstalk is vital to guide the rational combination of approved and experimental anticancer agents. Resistance to targeted therapies can result from de novo pathway redundancy, from acquired feedback activation of parallel pathways and from de novo or acquired cell plasticity. It is clear that metastatic cancers are increasingly heterogeneous, that is many different cell types and states are present within cancer tissues, and importantly metastatic cancers show increased resistance to radiation, chemotherapies, and targeted therapies. Cellular plasticity in epithelial cancers has been associated with progression and resistant to anticancer therapies. Several forms of plasticity have been documented, including epithelial mesenchymal transition (EMT), endothelial-mesenchymal transition, and neuroendocrine-epithelial transition. Epithelial mesenchymal transition (EMT) is an important cellular change promoting metastatic cell survival. We have shown mesenchymal-like tumors are relatively resistant to EGFR-TKi (Thomson, Cancer Res 2005), to IGF1R TKi (Buck, Cancer Res 2008), and to a broad spectrum of chemotherapy agents. A similar resistance of EMT-derived cells to anticancer therapies has been shown clinically in breast cancer patients (Creighton PNAS 2009), in prostate cancers (Domingo-Domenech, Cancer Cell 2012), and in NSCLC patients (Sequist, Cancer Cell 2012). Finally clinical EMT biomarkers predict response to EGFR inhibitors in NSCLC patients (Yauch, Clin. Cancer Res 2005; Byers et al., Clin Cancer Res 2012). New therapeutics are needed to target the new spectrum of tumor survival signals now present within EMT-derived cells and within related cancer-stem cells Through the generation and molecular characterization of EMT models, coupled with RNAi-based target validation studies and follow-on pharmacology studies, new targets and compounds which promote death of metastatic cells can be identified and validated in panels of cell lines and in animal models. Cell models for in vitro and in vivo target progression will broadly reflect both inducible and epigenetically-fixed mesenchymal cell states. In two cases (H358 and A549), TGFbeta was used to induce a reversible metastable mesenchymal cell state. The role of TGFbeta as both tumor suppressor and promoter of metastatic progression highlight the altered signal transduction cascades downstream of TGFbeta receptor activation in comparing epithelial and mesenchymal cell states. Here we show that mesenchymal-like tumor cells derived by EMT are preferentially sensitive to perturbations in integrin, focal adhesion and MAPK signaling pathways. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN06-03. Citation Format: John D. Haley. Cell plasticity promotes drug resistance and pathway redundancy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN06-03.

The mesenchymal stem cell potential of human dental pulp derived cells transfected with embryonic transcription factor Oct-4

Dental pulp stem cells (DPSCs) have shown promising potential in dental tissue repair and regeneration. However, the applications of DPSCs are hindered by obstacles such as the limited number of these cells within the dental pulp and the difficulty in purification of these cells. Therefore, it is necessary to develop a more efficient strategy by which DPSCs can be easily retrieved from dental pulp for tissue engineering applications. The embryonic transcription factor Oct-4 is often referred to as the master regulator of the undifferentiated state. Although its role in inducing pluripotent stem cells (iPS) is well established, its ability to directly reprogram postnatal dental pulp derived cells is not well defined. Using transient adenoviral-mediated transfection, we tested its ability to revert human dental pulp cells to a more mesenchymal stem cell-like phenotype. Results showed that Oct-4 transfected cells up-regulated expression of mesenchymal stem cells marker STRO-1, CD146, CD29 and CD44. Cells are able to advance into osteogenic, adipogenic and chondrogenic differentiation pathways. When loaded with ceramic scaffold and implanted subcutaneously in mice, only the cultures with Oct-4 gene transfection showed obvious bone-like hard tissue generation. These findings suggest that differentiated dental pulp cells can be reverted into mesenchymal stem cell state through the use of Oct-4 single factor transient transfection. This finding also has implications for easily acquiring of therapeutic cells from postnatal tissues for tissue engineering applications.

Paracrine and Autocrine Signals Induce and Maintain Mesenchymal and Stem Cell States in the Breast

The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of motility, invasiveness, and self-renewal traits. During both normal development and tumor pathogenesis, this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment. The signals that are responsible for inducing an EMT and maintaining the resulting cellular state have been unclear. We describe three signaling pathways, involving transforming growth factor (TGF)-β and canonical and noncanonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal state. Downregulation of endogenously synthesized inhibitors of autocrine signals in epithelial cells enables the induction of the EMT program. Conversely, disruption of autocrine signaling by added inhibitors of these pathways inhibits migration and self-renewal in primary mammary epithelial cells and reduces tumorigenicity and metastasis by their transformed derivatives.

MiR-200a Regulates Epithelial-Mesenchymal to Stem-like Transition via ZEB2 and β-Catenin Signaling

The emerging concept of generating cancer stem cells from epithelial-mesenchymal transition has attracted great interest; however, the factors and molecular mechanisms that govern this putative tumor-initiating process remain largely elusive. We report here that miR-200a not only regulates epithelial-mesenchymal transition but also stem-like transition in nasopharyngeal carcinoma cells. We first showed that stable knockdown of miR-200a promotes the transition of epithelium-like CNE-1 cells to the mesenchymal phenotype. More importantly, it also induced several stem cell-like traits, including CD133(+) side population, sphere formation capacity, in vivo tumorigenicity in nude mice, and stem cell marker expression. Consistently, stable overexpression of miR-200a switched mesenchyme-like C666-1 cells to the epithelial state, accompanied by a significant reduction of stem-like cell features. Furthermore, in vitro differentiation of the C666-1 tumor sphere resulted in diminished stem-like cell population and miR-200a induction. To investigate the molecular mechanism, we demonstrated that miR-200a controls epithelial-mesenchymal transition by targeting ZEB2, although it regulates the stem-like transition differentially and specifically by β-catenin signaling. Our findings reveal for the first time the function of miR-200a in shifting nasopharyngeal carcinoma cell states via a reversible process coined as epithelial-mesenchymal to stem-like transition through differential and specific mechanisms.

MicroRNAs and EMT in Mammary Cells and Breast Cancer

MicroRNAs are master regulators of gene expression in many biological and pathological processes, including mammary gland development and breast cancer. The differentiation program termed the epithelial to mesenchymal transition (EMT) involves changes in a number of microRNAs. Some of these microRNAs have been shown to control cellular plasticity through the suppression of EMT-inducers or to influence cellular phenotype through the suppression of genes involved in defining the epithelial and mesenchymal cell states. This has led to the suggestion that microRNAs maybe a novel therapeutic target for the treatment of breast cancer. In this review, we will discuss microRNAs that are involved in EMT in mammary cells and breast cancer.

Molecular mechanisms regulating chondroblast differentiation.

Formation of the cartilage template involves a multi-step process in which prechondrogenic mesenchymal cells form condensations prior to differentiating into matrix-producing chondroblasts. Retinoids, particularly retinoic acid, are among the numerous signaling molecules that have been implicated in this process. A proper balance of retinoids is essential for normal skeletal development in that too much or too little negatively impacts skeletogenesis. During the past few years, substantial advances have been made in our understanding of the role of retinoid signaling in these processes, which is reviewed in this report. To examine the function of retinoid signaling in skeletal development, transgenic mice that overexpressed a weak, constitutively active retinoic acid receptor (retinoic acid receptor-alpha) in their developing limbs were generated. The mice presented with a range of skeletal abnormalities. To examine the mechanisms responsible for these abnormalities, primary limb mesenchymal cultures from the transgenic mice were compared with cultures from wild-type mice. In addition, to address the molecular basis of retinoic acid receptor action, retinoic acid receptor activity in the primary cultures was manipulated with use of retinoic acid receptor-selective agonists and antagonists. The evaluation of the response to the manipulation of retinoic acid receptors was followed by histological studies and by the use of Northern blot analysis and reporter assays to analyze changes in the expression of chondrocytic markers and to monitor transcription factor activity, respectively. The evidence reviewed here indicates that retinoids maintain cells within condensations in a prechondrogenic, mesenchymal cell state, which prevents the cells from differentiating into chondroblasts. More recent studies have demonstrated that the inhibition of receptor-mediated retinoid signaling induces the expression of Sox9, a transcription factor that is considered a "master switch" for the differentiation of chondroblasts. These effects are largely mediated by the activation of the p38 MAPK signaling cascade. These findings demonstrate that retinoid receptor-mediated repression is both necessary and sufficient for chondroblast differentiation. Moreover, retinoic acid receptor repression acts downstream of BMP signaling or in a distinct pathway to activate p38 MAPK, which in turn induces chondroblast differentiation.

Serological identification of Streptococcus sanguis and Str mitior.

A total of 165 strains of Streptococcus sanguis and Str mitior were selected on the basis of their biochemical reactions using established identification procedures. These strains were also classified using API Database and were then screened against five candidate grouping sera. Biochemical tests and serological identification were in general complementary, but no regular associations between biotype and serological reaction were observed.

NEOPLASIA OF AORTIC INTIMA.

Two cases of arterial obstruction, in the region of the aortic bifurcation, are described. In both cases, cellular proliferation of neoplastic type was found to be causing the obstruction, and in one case small embolic metastases were found in the lower limbs and lungs. No primary tumour was found in any site other than the aortic intima itself. Two apparently similar cases have been reported in recent years. It is suggested that this condition, although probably very rare, may be overlooked for several reasons unless obstructive aortic lesions are examined histologically as a routine.