• Angiotensin II-Induced Protein Kinase D Activation and Regulation of Aldosterone Production

      Olala, Lawrence O.; Department of Pathology (2013-02)
      Dysregulated aldosterone production leading to hypertension and its associated complications, such as congestive heart failure, cardiac fibrosis and renal failure, are important public health concerns with a huge impact on the economy and patient quality of life. Thus, there is a high level of interest in the development of medical interventions and lifestyle changes to reduce the incidence of hypertension. Stimulation of the adrenal zona glomerulosa with angiotensin II (AngII), potassium (K+) or adrenocorticotropic hormone (ACTH), increases aldosterone production, to result in increased sodium and water retention. We have recently shown a role for the serine/threonine protein kinase D (PKD) in the regulation of acute aldosterone synthesis upon AngII stimulation. In this study, using both molecular and pharmacological approaches, we demonstrate that Src family kinases and protein kinase C (PKC) activate PKD to increase aldosterone production in bovine adrenal glomerulosa cells. We have also shown that PKD positively regulates expression of steroidogenic acute regulatory (StAR) protein, a protein required for cholesterol transport into the mitochondria, and aldosterone synthesis. PKD plays this role, in part, through activating members of the activating transcription factor (ATF)/cAMP response element (CRE-) binding protein (CREB) family of leucine zipper transcription factors. Therefore, we hypothesize that PKC and Src family kinase-mediated PKD activation in response to AngII increases the phosphorylation and activation of ATF-2 and CREB, which bind the StAR proximal promoter thereby resulting in induction of StAR expression and stimulation of steroidogenesis.
    • Aortic Calcification and Femoral Bone Density Are Independently Associated with Left Ventricular Mass in Patients with Chronic Kidney Disease

      Chue, Colin D.; Wall, Nadezhda A.; Crabtree, Nicola J.; Zehnder, Daniel; Moody, William E.; Edwards, Nicola C.; Steeds, Richard P.; Townend, Jonathan N.; Ferro, Charles J.; Shi, Xing-Ming; et al. (2012-06-18)
      Background: Vascular calcification and reduced bone density are prevalent in chronic kidney disease and linked to increased cardiovascular risk. The mechanism is unknown. We assessed the relationship between vascular calcification, femoral bone density and left ventricular mass in patients with stage 3 non-diabetic chronic kidney disease in a cross-sectional observational study.
    • Consensus nomenclature for the human ArfGAP domain-containing proteins

      Kahn, Richard A.; Bruford, Elspeth; Inoue, Hiroki; Logsdon, John M.; Nie, Zhongzhen; Premont, Richard T.; Randazzo, Paul A.; Satake, Masanobu; Theibert, Anne B.; Zapp, Maria L.; et al. (2008-09-22)
      At the FASEB summer research conference on â Arf Family GTPasesâ , held in Il Ciocco, Italy in June, 2007, it became evident to researchers that our understanding of the family of Arf GTPase activating proteins (ArfGAPs) has grown exponentially in recent years. A common nomenclature for these genes and proteins will facilitate discovery of biological functions and possible connections to pathogenesis. Nearly 100 researchers were contacted to generate a consensus nomenclature for human ArfGAPs. This article describes the resulting consensus nomenclature and provides a brief description of each of the 10 subfamilies of 31 human genes encoding proteins containing the ArfGAP domain.
    • Cortical Thickness Mapping to Identify Focal Osteoporosis in Patients with Hip Fracture

      Poole, Kenneth E.S.; Treece, Graham M.; Mayhew, Paul M.; Vaculí­k, Jan; Dungl, Pavel; Horák, Martin; Štӗpán, Jan J.; Gee, Andrew H.; Shi, Xing-Ming; Department of Pathology (2012-06-11)
      Background: Individuals with osteoporosis are predisposed to hip fracture during trips, stumbles or falls, but half of all hip fractures occur in those without generalised osteoporosis. By analysing ordinary clinical CT scans using a novel cortical thickness mapping technique, we discovered patches of markedly thinner bone at fracture-prone regions in the femurs of women with acute hip fracture compared with controls.
    • Dynamin2- and endothelial nitric oxide synthaseâ regulated invasion of bladder epithelial cells by uropathogenic Escherichia coli

      Wang, Zhimin; Humphrey, Ceba; Frilot, Nicole; Wang, Gaofeng; Nie, Zhongzhen; Moniri, Nader H.; Daaka, Yehia; Department of Pathology (2011-01-10)
      Invasion of bladder epithelial cells by uropathogenic Escherichia coli (UPEC) contributes to antibiotic-resistant and recurrent urinary tract infections (UTIs), but this process is incompletely understood. In this paper, we provide evidence that the large guanosine triphosphatase dynamin2 and its partner, endothelial nitric oxide (NO) synthase (NOS [eNOS]), mediate bacterial entry. Overexpression of dynamin2 or treatment with the NO donor S-nitrosothiols increases, whereas targeted reduction of endogenous dynamin2 or eNOS expression with ribonucleic acid interference impairs, bacterial invasion. Exposure of mouse bladder to small molecule NOS inhibitors abrogates infection of the uroepithelium by E. coli, and, concordantly, bacteria more efficiently invade uroepithelia isolated from wild-type compared with eNOSâ /â mice. E. coli internalization promotes rapid phosphorylation of host cell eNOS and NO generation, and dynamin2 S-nitrosylation, a posttranslational modification required for the bacterial entry, also increases during E. coli invasion. These findings suggest that UPEC escape urinary flushing and immune cell surveillance by means of eNOS-dependent dynamin2 S-nitrosylation and invasion of host cells to cause recurrent UTIs.
    • The Early Stage Adjacent Disc Degeneration after Percutaneous Vertebroplasty and Kyphoplasty in The Treatment of Osteoporotic VCFs

      Qian, Jun; Yang, Huilin; Jing, Juehua; Zhao, Hong; Ni, Li; Tian, Dasheng; Wang, Zhengfei; Shi, Xing-Ming; Department of Pathology; College of Graduate Studies (2012-10-8)
      Background: The purpose of this paper is to determine the early incidence of disc de- generation adjacent to the vertebral body of osteoporotic fracture treated with percutaneous vertebroplasty or balloon kyphoplasty and whether adjacent disc degeneration is accelerated by this two procedures.
    • Heat shock response in CHO mammalian cells is controlled by a nonlinear stochastic process.

      Lipan, Ovidiu; Navenot, Jean-Marc; Wang, Zixuan; Huang, Lei; Peiper, Stephen C; Department of Pathology; GHSU Cancer Center; Immunotherapy Center; Department of Radiology; Center for Molecular Chaperone/Radiobiology & Cancer Virology (2007-10-30)
      In many biological systems, the interactions that describe the coupling between different units in a genetic network are nonlinear and stochastic. We study the interplay between stochasticity and nonlinearity using the responses of Chinese hamster ovary (CHO) mammalian cells to different temperature shocks. The experimental data show that the mean value response of a cell population can be described by a mathematical expression (empirical law) which is valid for a large range of heat shock conditions. A nonlinear stochastic theoretical model was developed that explains the empirical law for the mean response. Moreover, the theoretical model predicts a specific biological probability distribution of responses for a cell population. The prediction was experimentally confirmed by measurements at the single-cell level. The computational approach can be used to study other nonlinear stochastic biological phenomena.
    • Intraoperative neurocytology of primary central nervous system neoplasia: A simplified and practical diagnostic approach

      Sharma, Suash; Deb, Prabal; Department of Pathology (2011)
      Intraoperative consultations may pose considerable diagnostic challenge to the neuropathologist in diagnosing primary and metastatic neoplasms of the central nervous system (CNS). Cytological preparations in the form of squash, touch, imprint or smears are few of the available modalities in addition to the frozen section (FS). Although the latter is superior in providing both histologic patterns and cytomorphologic details yet smears are of vital importance when tissue available is limited (stereotactic biopsy), scrutinisation of intercellular matrix (astrocytoma versus oligodendroglioma) and evaluation of discohesive cells (lymphoma, pituitary adenoma) and in inflammatory lesions. This review is intended to emphasize the value, applicability and limitations of neurocytology aiming to expedite the intraoperative smear-based diagnoses of CNS neoplasia as per the World Health Organization (WHO) classification. We recommend that whenever possible, both smears and FS should be examined concomitantly and in a correlative manner. In the unlikely event of a mismatch between the findings on smear and FS, intraoperative diagnosis is primarily based on FS, if adequate tissue is available. However, each case must be evaluated on its own merit and in difficult cases relevant differential diagnoses should be offered to facilitate surgical decisions and optimally triage patient management.
    • Involvement of p54(nrb), a PSF partner protein, in DNA double-strand break repair and radioresistance.

      Li, Shuyi; Kuhne, Wendy W.; Kulharya, Anita; Hudson, Farlyn Z; Ha, Kyungsoo; Cao, Zhen; Dynan, William S.; Department of Pathology (2009-11-16)
      Mammalian cells repair DNA double-strand breaks (DSBs) via efficient pathways of direct, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Prior work has identified a complex of two polypeptides, PSF and p54(nrb), as a stimulatory factor in a reconstituted in vitro NHEJ system. PSF also stimulates early steps of HR in vitro. PSF and p54(nrb) are RNA recognition motif-containing proteins with well-established functions in RNA processing and transport, and their apparent involvement in DSB repair was unexpected. Here we investigate the requirement for p54(nrb) in DSB repair in vivo. Cells treated with siRNA to attenuate p54(nrb) expression exhibited a delay in DSB repair in a gamma-H2AX focus assay. Stable knockdown cell lines derived by p54(nrb) miRNA transfection showed a significant increase in ionizing radiation-induced chromosomal aberrations. They also showed increased radiosensitivity in a clonogenic survival assay. Together, results indicate that p54(nrb) contributes to rapid and accurate repair of DSBs in vivo in human cells and that the PSF.p54(nrb) complex may thus be a potential target for radiosensitizer development.
    • Isolation of Osteoprogenitors from Human Jaw Periosteal Cells: A Comparison of Two Magnetic Separation Methods

      Olbrich, Marcus; Rieger, Melanie; Reinert, Siegmar; Alexander, Dorothea; Department of Pathology; College of Graduate Studies (2012-10-19)
      Human jaw periosteum tissue contains osteoprogenitors that have potential for tissue engineering applications in oral and maxillofacial surgeries. To isolate osteoprogenitor cells from heterogeneous cell populations, we used the specific mesenchymal stem cell antigen-1 (MSCA-1) antibody and compared two magnetic separation methods. We analyzed the obtained MSCA-1+ and MSCA-1â fractions in terms of purity, yield of positive/negative cells and proliferative and mineralization potentials. The analysis of cell viability after separation revealed that the EasySep method yielded higher viability rates, whereas the flow cytometry results showed a higher purity for the MACS-separated cell fractions. The mineralization capacity of the osteogenic induced MSCA-1+ cells compared with the MSCA-1â controls using MACS was 5-fold higher, whereas the same comparison after EasySep showed no significant differences between both fractions. By analyzing cell proliferation, we detected a significant difference between the proliferative potential of the osteogenic cells versus untreated cells after the MACS and EasySep separations. The differentiated cells after MACS separation adjusted their proliferative capacity, whereas the EasySep-separated cells failed to do so. The protein expression analysis showed small differences between the two separation methods. Our findings suggest that MACS is a more suitable separation method to isolate osteoprogenitors from the entire jaw periosteal cell population.
    • A modified hyperplane clustering algorithm allows for efficient and accurate clustering of extremely large datasets.

      Sharma, Ashok; Podolsky, Robert H.; Zhao, Jieping; McIndoe, Richard A; Department of Pathology (2009-04-24)
      MOTIVATION: As the number of publically available microarray experiments increases, the ability to analyze extremely large datasets across multiple experiments becomes critical. There is a requirement to develop algorithms which are fast and can cluster extremely large datasets without affecting the cluster quality. Clustering is an unsupervised exploratory technique applied to microarray data to find similar data structures or expression patterns. Because of the high input/output costs involved and large distance matrices calculated, most of the algomerative clustering algorithms fail on large datasets (30,000 + genes/200 + arrays). In this article, we propose a new two-stage algorithm which partitions the high-dimensional space associated with microarray data using hyperplanes. The first stage is based on the Balanced Iterative Reducing and Clustering using Hierarchies algorithm with the second stage being a conventional k-means clustering technique. This algorithm has been implemented in a software tool (HPCluster) designed to cluster gene expression data. We compared the clustering results using the two-stage hyperplane algorithm with the conventional k-means algorithm from other available programs. Because, the first stage traverses the data in a single scan, the performance and speed increases substantially. The data reduction accomplished in the first stage of the algorithm reduces the memory requirements allowing us to cluster 44,460 genes without failure and significantly decreases the time to complete when compared with popular k-means programs. The software was written in C# (.NET 1.1). AVAILABILITY: The program is freely available and can be downloaded from http://www.amdcc.org/bioinformatics/bioinformatics.aspx. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    • Ontological Differences in First Compared to Third Trimester Human Fetal Placental Chorionic Stem Cells

      Jones, Gemma N.; Moschidou, Dafni; Puga-Iglesias, Tamara-Isabel; Kuleszewicz, Katarzyna; Vanleene, Maximilien; Shefelbine, Sandra J.; Bou-Gharios, George; Fisk, Nicholas M.; David, Anna L.; De Coppi, Paolo; et al. (2012-09-4)
      Human mesenchymal stromal/stem cells (MSC) isolated from fetal tissues hold promise for use in tissue engineering applications and cell-based therapies, but their collection is restricted ethically and technically. In contrast, the placenta is a potential source of readily-obtainable stem cells throughout pregnancy. In fetal tissues, early gestational stem cells are known to have advantageous characteristics over neonatal and adult stem cells. Accordingly, we investigated whether early fetal placental chorionic stem cells (e-CSC) were physiologically superior to their late gestation fetal chorionic counterparts (l-CSC). We showed that e-CSC shared a common phenotype with l-CSC, differentiating down the osteogenic, adipogenic and neurogenic pathways, and containing a subset of cells endogenously expressing NANOG, SOX2, c-MYC, and KLF4, as well as an array of genes expressed in pluripotent stem cells and primordial germ cells, including CD24, NANOG, SSEA4, SSEA3, TRA-1-60, TRA-1-81, STELLA, FRAGILIS, NANOS3, DAZL and SSEA1. However, we showed that e-CSC have characteristics of an earlier state of stemness compared to l-CSC, such as smaller size, faster kinetics, uniquely expressing OCT4A variant 1 and showing higher levels of expression of NANOG, SOX2, c-MYC and KLF4 than l-CSC. Furthermore e-CSC, but not l-CSC, formed embryoid bodies containing cells from the three germ layer lineages. Finally, we showed that e-CSC demonstrate higher tissue repair in vivo; when transplanted in the osteogenesis imperfecta mice, e-CSC, but not l-CSC increased bone quality and plasticity; and when applied to a skin wound, e-CSC, but not l-CSC, accelerated healing compared to controls. Our results provide insight into the ontogeny of the stemness phenotype during fetal development and suggest that the more primitive characteristics of early compared to late gestation fetal chorionic stem cells may be translationally advantageous.
    • ParaSAM: a parallelized version of the significance analysis of microarrays algorithm.

      Sharma, Ashok; Zhao, Jieping; Podolsky, Robert H.; McIndoe, Richard A; Department of Pathology (2010-05-20)
      MOTIVATION: Significance analysis of microarrays (SAM) is a widely used permutation-based approach to identifying differentially expressed genes in microarray datasets. While SAM is freely available as an Excel plug-in and as an R-package, analyses are often limited for large datasets due to very high memory requirements. SUMMARY: We have developed a parallelized version of the SAM algorithm called ParaSAM to overcome the memory limitations. This high performance multithreaded application provides the scientific community with an easy and manageable client-server Windows application with graphical user interface and does not require programming experience to run. The parallel nature of the application comes from the use of web services to perform the permutations. Our results indicate that ParaSAM is not only faster than the serial version, but also can analyze extremely large datasets that cannot be performed using existing implementations. AVAILABILITY: A web version open to the public is available at http://bioanalysis.genomics.mcg.edu/parasam. For local installations, both the windows and web implementations of ParaSAM are available for free at http://www.amdcc.org/bioinformatics/software/parasam.aspx.
    • Pediatric primary intramedullary spinal cord glioblastoma

      Lober, Robert M.; Sharma, Suash; Bell, Beverly; Free, Alan; Figueroa, Ramon; Sheils, Chris W; Lee, Mark R.; Cowell, John K.; Department of Pathology; Department of Pediatrics; et al. (2010-09-30)
      Spinal cord tumors in pediatric patients are rare, representing less than 1% of all central nervous system tumors. Two cases of pediatric primary intramedullary spinal cord glioblastoma at ages 14 and 8 years are reported. Both patients presented with rapid onset paraparesis and quadraparesis. Magnetic resonance imaging in both showed heterogeneously enhancing solitary mass lesions localized to lower cervical and upper thoracic spinal cord parenchyma. Histopathologic diagnosis was glioblastoma. Case #1 had a small cell component (primitive neuroectodermal tumor-like areas), higher Ki67, and p53 labeling indices, and a relatively stable karyotype with only minimal single copy losses involving regions: Chr8;pter-30480019, Chr16;pter-29754532, Chr16;56160245â 88668979, and Chr19;32848902-qter on retrospective comparative genomic hybridization using formalin-fixed, paraffin-embedded samples. Case #2 had relatively bland histomorphology and negligible p53 immunoreactivity. Both underwent multimodal therapy including gross total resection, postoperative radiation and chemotherapy. However, there was no significant improvement in neurological deficits, and overall survival in both cases was 14 months.This report highlights the broad histological spectrum and poor overall survival despite multi modality therapy. The finding of relatively unique genotypic abnormalities resembling pediatric embryonal tumors in one case may highlight the value of genome-wide profiling in development of effective therapy. The differences in management with intracranial and low-grade spinal cord gliomas and current management issues are discussed.
    • Primary mucinous adenocarcinoma in a defunctionalized urinary bladder: a case report.

      Taneous, Mary; Ramalingam, Preetha; Mode, Donald G; Heiner, Jared G; Terris, Martha K; Lee, Jeffrey R.; Department of Pathology; Department of Surgery (2010-01-11)
      INTRODUCTION: Malignancies are rare in defunctionalized bladders and are thought to arise from metaplasia secondary to chronic inflammation. Transitional cell and squamous cell carcinomas are the most common but there are three reported cases of mucinous adenocarcinoma. CASE PRESENTATION: We report a 57-year-old Caucasian man presenting with penile discharge for 30 years following ileal conduit surgery for neurogenic bladder, and who was found to have primary mucinous adenocarcinoma of his defunctionalized bladder. CONCLUSION: Although urinary diversion without cystectomy is less common in current urologic practice, there are many patients with longstanding defunctionalized bladders. While there are no established surveillance protocols, defunctionalized bladder patients with urethral discharge should be evaluated.
    • Switched alternative splicing of oncogene CoAA during embryonal carcinoma stem cell differentiation.

      Yang, Zheqiong; Sui, Yang; Xiong, Shiqin; Liour, Sean S; Phillips, Andrew C; Ko, Lan; Department of Pathology (2007-04-23)
      Alternative splicing produces functionally distinct proteins participating in cellular processes including differentiation and development. CoAA is a coactivator that regulates transcription-coupled splicing and its own pre-mRNA transcript is alternatively spliced. We show here that the CoAA gene is embryonically expressed and alternatively spliced in multiple tissues to three splice variants, CoAA, CoAM and CoAR. During retinoic-acid-induced P19 stem cell differentiation, the expression of CoAA undergoes a rapid switch to its dominant negative splice variant CoAM in the cavity of the embryoid body. CoAM functionally inhibits CoAA, and their switched expression up-regulates differentiation marker Sox6. Using a CoAA minigene cassette, we find that the switched alternative splicing of CoAA and CoAM is regulated by the cis-regulating sequence upstream of the CoAA basal promoter. Consistent to this, we show that p54(nrb) and PSF induce CoAM splice variant through the cis-regulating sequence. We have previously shown that the CoAA gene is amplified in human cancers with a recurrent loss of this cis-regulating sequence. These results together suggest that the upstream regulatory sequence contributes to alternative splicing of the CoAA gene during stem cell differentiation, and its selective loss in human cancers potentially deregulates CoAA alternative splicing and alters stem cell differentiation.
    • Transcriptional Coactivator and Oncoprotein CoAA

      Brooks, Yang Sui; Department of Pathology (2008)
      CoAA contains two copies of RNA recognition motifs (RRM) and an intrinsic transactivation domain rich in repetitive tyrosines and glutamines (YxxQ domain). Previously, CoAA has been shown to be a transcriptional coactivator that stimulates transcriptional activation and regulates alternative splicing. A pattern and profile search revealed that the YxxQ domain in CoAA shared significant pattern homology with the oncogenic EWS activation domains (EAD) in TET family proteins, including, TLS/FUS, EWS and TAFII 68. It was further demonstrated that CoAA’s YxxQ domain and EWS’ EAD also shared functional similarities. Based on these findings, this work investigated the aberration of CoAA in cancers and its pathophysiological significance. The results showed that the CoAA gene was amplified in a high percentage of inflammation-related human cancers with recurrent loss of the 5’ regulatory element upstream of its promoter. This genomic aberration resulted in CoAA protein overexpression, which in turn, induced the transformation of NIH3T3 cells. Subsequently, it was shown that the lost 5’ regulatory element could modulate the alternative splicing of the CoAA gene during stem cell differentiation and that the unbalanced expression of CoAA and its splice variant, CoAM could potentially impact the cell differentiation process. To further characterize the regulation of CoAA alternative splicing, two conserved trans-splicing events between CoAA and its downstream RBM4 were identified. These events yield a novel zinc finger- containing coactivator, CoAZ, and a non-coding splice variant, ncCoAZ. Both variants regulated their parental genes’ mRNA expression as well as activities, suggesting a linked control between CoAA and RBM4. Moreover, the expression patterns of CoAA, RBM4 and their trans-splicing variants switched during neural stem cell differentiation, resulting in lineage-specific expression of each variant. Our phylogenetic analysis suggests that mammalian CoAA and RBM4 share a common ancestor with the Drosophila melanogaster gene, Lark. In this regard, the trans-splicing events between CoAA and RBM4 represent a functional regulation preserved during evolution. This study established the connection between CoAA and human cancer and provides evidence for CoAA’s involvement in the regulation of cell differentiation. Moreover, this study is the first to report a functional trans-splicing variant in mammalian cells.
    • The Transcriptional Profile of Mesenchymal Stem Cell Populations in Primary Osteoporosis Is Distinct and Shows Overexpression of Osteogenic Inhibitors

      Benisch, Peggy; Schilling, Tatjana; Klein-Hitpass, Ludger; Frey, Sonke P.; Seefried, Lothar; Raaijmakers, Nadja; Krug, Melanie; Regensburger, Martina; Zeck, Sabine; Schinke, Thorsten; et al. (2012-09-24)
      Primary osteoporosis is an age-related disease characterized by an imbalance in bone homeostasis. While the resorptive aspect of the disease has been studied intensely, less is known about the anabolic part of the syndrome or presumptive deficiencies in bone regeneration. Multipotent mesenchymal stem cells (MSC) are the primary source of osteogenic regeneration. In the present study we aimed to unravel whether MSC biology is directly involved in the pathophysiology of the disease and therefore performed microarray analyses of hMSC of elderly patients (79â 94 years old) suffering from osteoporosis (hMSC-OP). In comparison to age-matched controls we detected profound changes in the transcriptome in hMSC-OP, e.g. enhanced mRNA expression of known osteoporosis-associated genes (LRP5, RUNX2, COL1A1) and of genes involved in osteoclastogenesis (CSF1, PTH1R), but most notably of genes coding for inhibitors of WNT and BMP signaling, such as Sclerostin and MAB21L2. These candidate genes indicate intrinsic deficiencies in self-renewal and differentiation potential in osteoporotic stem cells. We also compared both hMSC-OP and non-osteoporotic hMSC-old of elderly donors to hMSC of â ¼30 years younger donors and found that the transcriptional changes acquired between the sixth and the ninth decade of life differed widely between osteoporotic and non-osteoporotic stem cells. In addition, we compared the osteoporotic transcriptome to long term-cultivated, senescent hMSC and detected some signs for pre-senescence in hMSC-OP.
    • Transcriptional Regulation by Tbx2

      Chen, Jung-Ren; Department of Pathology (2001-03)
      Tbx2 is a member of an evolutionarily conserved transcriptional regulatory gene family. Little is known about the molecular mechanisms underlying the function of Tbx2. Because connexin43 (Cx43) and Tbx2 are both expressed in neural crest derivatives in pharyngeal arches and because the promoter of Cx43 contains direct repeats of T (Brachyury) half sites, it is hypothesized that Tbx2 regulates Cx43 and other genes important for neural crest cell functions. TBX2 DNA binding affinity was analyzed by eletrophoretic mobility shift assays. Transcriptional regulation of the Cx43 promoter by Tbx2 was analyzed using reporter constructs. These results suggest that Cx43 is a bona fide target gene o(Tbx2 and that Tbx2 negatively regulates Cx43 gene expression by binding to TCACAC sites. Moreover, dye-coupling assays showed that Tbx2 upregulation led to decreased junctional coupling. To identify other genes that may be regulated by Tbx2, a differential gene expression profile was determined using GEM1 microarrays. CellSpace knowledgebase was used to perform functional assignments to 72>x2-regulated genes. This analysis indicated that Tbx2 might be involved in different fundamental cell functions. Tbx2 upregulates proliferation genes, downregulates tenascinC, and upregulates nidogen. In conclusion, together with Cx43 repression, Tbx2 may signal neural crest cells to stop migration and start proliferation. Gene cluster analysis also suggested a potential role for Tbx2 in osteogenesis. In accord, Tbx2 expression was detected in chondrocytes and osteocytes both in long bone and membranous bones.