The IVASC section held our annual student and post doc competition. Each student and post doc presented an oral talk lasting approximately 15 minutes. We had three competitors this year. Each participant gave an excellent talk which generated lots of questions. A cash award was given for first, second and third place. First place, went to Sung-Yong Hwang, second place to Anna De La Fuente, and third place to Vinita Daniel. We extend congratulations to all the participants and thanks to our judges, John Harbell, Bill Smith and Lia Campbell.

Submitted by Lia Campbell

Sung-Yong Hwang

First place:

Orai1-Mediated Calcium Entry Regulates Differentiation and Function of Osteoclast Cells: Implications in Drug Development for Bone Diseases such as Osteoporosis and Rheumatoid Arthritis

Osteoclasts are bone resorbing cells formed by the fusion of bone marrow-derived monocyte/macrophage cells (BMMs). Osteoclasts attach to the bone surface and resorb bone by secreting acid and bone degrading enzymes. Excessive bone resorption by osteoclasts results in a number of bone diseases such as postmenopausal osteoporosis and rheumatoid arthritis. Receptor activator of nuclear factor kappa B ligand (RANKL) acts as an initial signal for osteoclastogenesis. It has been shown that NFATc1, a nuclear transcription factor, is selectively induced during RANKL-mediated osteoclastogenesis and plays as a master regulator of osteoclast differentiation. NFATc1 is activated by calcium signaling initiated by RANKL. However, the calcium channel responsible for the calcium signaling remains elusive. The contribution of Orai1, a recently identified store-operated calcium channel, to the calcium signaling of osteoclasts has not been investigated. Using a gene silencing technique, we found that knockdown of Orai1 by lentivirus expressing Orai1 shRNA suppressed store-operated calcium entry and osteoclastogenesis of RAW264.7 via impairment of multinucleation of osteoclast cells. The functional resorbing activity was also diminished in the osteoclasts infected with Orai1 shRNA virus compared to the control group. As a possible mechanism by which Orai1 regulates osteoclast differentiation, we found by real-time RT-PCR that induction of NFATc1 was significantly blunted in the osteoclasts infected with Orai1 shRNA virus. Furthermore, Western blot analysis demonstrated that knockdown of Orai1 suppressed the calcium dependent nuclear translocation of NFATc1, leading to concomitant inhibition of downstream NFATc1-targeted genes essential for osteoclastogenesis. In the present study we show that Orai1-mediated calcium entry plays a critical role in osteoclast differentiation and function via NFATc1-dependent gene induction. This may suggests that Orai1 could be a potential therapeutic target for the treatment or prevention of bone loss caused by osteoclasts.

Sung-Yong Hwang, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709. In Vitro Cellular and Developmental Biology, 47:S32, 2011

Second Place:

Anna de La Fuente

Identification of Cadmium-regulated miRNAs in Rat Renal Proximal Tubule Epithelial NRK-52E Cells

MicroRNAs (miRNAs) are small non-coding RNAs that function as post-transcriptional regulators of gene expression. Cadmium (Cd) is a major nephrotoxic environmental pollutant that selectively damages the proximal tubule. The objective of the present study was to examine the effect of Cd on miRNA expression in rat kidney proximal tubule epithelial NRK-52E cells to determine their potential involvement in Cd-induced nephrotoxicity. The NKR-52E cells were exposed to 0-10 µM CdCl2 in a serum-containing DMEM media for 24 hours. MicroRNAs were extracted using the mirVana microRNA isolation kit and analyzed using a rat miRNA LC Sciences microarray. The results showed that treatment of cells with 5 µM of CdCl2 causes phenotypic cellular alterations such as cell separation without causing overt cellular toxicity, as assessed by trypan blue and WST-1 assay. The results of the microRNA microarray analysis showed that acute exposure to CdCl2 significantly down-regulated rno-miR-30c and rno-miR-26a (t-test, p < 0.05), rno-miR-125a-5p and rno-miR-107 (p < 0.1), and significantly up-regulated rno-miR-466b-2, rno-miR-3584-5p, rno-miR-29a (p < 0.1). Nineteen additional miRNAs appeared to be altered following exposure to CdCl2 but low signal intensity precluded more detailed analysis. The present research indicates that exposure to non-cytotoxic concentrations of CdCl2 affects the expression of miRNAs in NRK-52E cells and raises the possibility that alteration in levels of miRNAs may play a role in Cd-induced proximal tubule injury.

Anna de La Fuente, Midwestern University, 555 31st St., Downers Grove, IL 60515. In Vitro Cellular and Developmental Biology, 47:S31-32, 2011

Third Place:

Vinita C. Daniel

Cullin-5 Knock-down Affects Gene Expression in Human Breast Cancer Cells

Breast cancer is the second leading cause of cancer-related death in women in the United States. The Cullin-5 (Cul5) gene is thought to play a role as a tumor suppressor in the development of breast cancer since breast cancer tissue commonly demonstrates decreased expression versus matched normal tissue. Cul5 is known to function in the ubiquitin-mediated protein degradation pathway; however, the tumor suppressor mechanisms are not well understood. To investigate the tumor suppressor roles of Cul5, an RNAi model of Cul5 knock-down using siRNAs and the MDA-MB-231 breast cancer cell line was used to screen for changes in gene expression using microarrays. A negative control or Cul5 siRNA were introduced into the cells using reverse transfection or electroporation, and knock-down was monitored using RT-PCR and Western blot analysis. Total RNA from the siRNA treated cells was used to screen for changes in gene expression using Human Breast Cancer and Estrogen Receptor, Human Tumor Metastasis, Human Cell Cycle and Human Apoptosis RT2 Profiler™ PCR Arrays. Cul5 knock-down was confirmed at the mRNA level in both electroporated and transfected cells as evidenced by the 80.4% and 58.7% decrease in Cul5 mRNA expression, respectively. Cul5 protein knock-down was also documented in siRNA transfected cells. Genes that were up or down regulated (≥1.5 fold) in a consistent manner with the two siRNA delivery methods were considered for future validation. Genes that were up regulated include MUC1 and KISS1. Genes that were down regulated include CIDEA, CD70, TNFSF8, LTA, CASP3, IL6, IL1B and CLU. The identification of genes that are differentially regulated in cancer cells with decreased expression of Cul5 may provide insight into the tumor suppressor functions of Cul5 in breast cancer.

Vinita C. Daniel, Midwestern University, 555 31st St., Downers Grove, IL 60515. In Vitro Cellular and Developmental Biology, 47:S31, 2011.

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