Bong Cho Kim

LDL stimulates collagen mRNA synthesis in mesangial cells through induction of PKC and TGF-β expression

Abnormal lipid accumulation in glomeruli could be implicated in the pathogenesis of glomerulosclerosis. Low-density lipoprotein (LDL) stimulates collagen mRNA expression in cultured human mesangial cells (HMC). To explore the possible molecular mechanisms by which LDL promotes collagen gene expression, we examined the effects of LDL on protein kinase C (PKC) activity and transforming growth factor-β (TGF-β) expression in relation to collagen gene regulation in HMC. LDL (200 μg/ml) induced an acute increase in PKC activity, particularly PKC-α and -δ, within 15 min, which decreased to control value at 2 h. LDL stimulated TGF-β1, and α1(I) and α1(IV) collagen mRNA expression within 30 min of incubation with HMC, and levels remained elevated until hour 4. LDL induced the secretion of TGF-β by HMC. This TGF-β was shown by CCL-64 mink lung cell assay to be, in part, bioactive. The stimulatory effects of LDL on collagen gene regulation in HMC were blocked by the inhibition of PKC using GF-109203X (GFX) or the downregulation of PKC using phorbol myristate acetate. Neutralizing antibody to TGF-β inhibited the increased collagen mRNA expression by HMC exposed to LDL. The downregulation or inhibition of PKC did not affect the stimulatory effect of LDL on TGF-β mRNA or protein expression. These results suggest that in HMC, LDL stimulates collagen mRNA expression through the rapid activation of PKC-α and -δ and transcriptional upregulation of TGF-β. Thus PKC and TGF-β may function as independent key signaling intermediaries in the pathway by which LDL upregulates collagen gene expression in HMC.

Glycated Albumin Induces Superoxide Generation in Mesangial Cells

Background/Aims: Reactive oxygen species are involved in the pathogenesis of diabetic nephropathy. Amadori-modified glycated albumin modulates signaling pathways in mesangial cells that contribute to the development of diabetic nephropathy. However, the effects of glycated albumin on mesangial cell superoxide (O₂^-) production are unknown. Thus, we examined whether glycated albumin induces mesangial cell O₂^- generation and whether increased O₂^- production elicits cell growth. Methods: Quiescent human mesangial cells (HMC) were exposed to bovine serum albumin (BSA) or glycated BSA (Gly-BSA) with or without diphenylene iodonium (DPI) or apocynin, inhibitors of NAD(P)H oxidase, GF109203X (GFX), a protein kinase C (PKC) inhibitor. Results: Gly-BSA increased PKC activity, particularly PKC-α and -α1, within 15 min of incubation with HMC, which decreased to the control value at 2 h. Gly-BSA incubated with HMC increased O₂^- production by 2 times vis-à-vis BSA-treated cells. The Gly-BSA-induced increased O₂^- generation was suppressed by DPI or GFX. Gly-BSA significantly increased mesangial [³H]-leucine incorporation, whereas these processes were abrogated by DPI, apocynin or GFX. Conclusions: Gly-BSA induces PKC/NAD(P)H oxidase-dependent O₂^- production in HMC, which in turn results in cell hypertrophy. Thus, O₂^- induced by glycated albumin might cause mesangial cell alterations in diabetes participating in the pathophysiology of diabetic nephropathy.

Abstract 3574: Analysis of whole genome and transcriptome sequencing in single cell

The rapid advancement in Next Generation Sequencing (NGS) technology has allowed extensive genetic profiling and analysis of tumor tissues. While accurate tumor characterization is crucial for cancer therapy, current sequencing methods are unable to resolve the genetic differences between the heterogeneous cell populations within tumor. By profiling specific subpopulations of cells from a larger heterogeneous population, single cell sequencing can improve the efficiency of cancer treatments through early detection of rare tumor cells, monitoring of circulating tumor cells, and accurate profiling of intra-tumor heterogeneity. In this study, we compared commercially available single cell amplification methods and sequencing platforms for whole genome and whole transcriptome sequencing. We isolated single cells from a human breast cancer cell line, MCF7, and human embryonic kidney cell line, HEK293, by micromanipulation, and their whole genome and transcriptome were sequenced. For whole genome sequencing, the performance of Repli-g (Qiagen), Ampli1 (Slicon Biosystems) and WGA4 (Sigma) were assessed for whole genome amplification. Next, we used TruSeq DNA Library Kit and Ion Plus Fragment Library Kit for library preparation, and whole genome sequencing was performed on HiSeq2000 and Proton. The sequencing data were analyzed by SAMtools/GATK and tmap-f3. For whole transcriptome sequencing, cDNA was synthesized from single cells using SMARTer ultra low input RNA kit (Clontech), and the cDNA library was prepared with Nextera XT library prep kit (Illumina) and Ion Total RNA-Seq Kit v2 (Life Technologies). Sequencing was performed on HiSeq2000 and Proton, and analyzed with Tophat and Cufflink. We show that the percentage of mappable reads and regions with more than 1X coverage in whole genome sequencing were similar to that of the genomic DNA data, but the coverage rate of regions with more than 10X was lower in single cell data on both NGS platforms. In the RNA-Seq, the gene detection rate in single cell and more than 100 cells were about 65% and 85% compare to total RNA, respectively. Citation Format: Nak-Jung Kwon, Woo Chung Lee, Jiwoong Kim, Hyeri Kim, Ahreum Seong, Bong Cho Kim, Doo Hyun Park, Kap-Seok Yang. Analysis of whole genome and transcriptome sequencing in single cell. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3574. doi:10.1158/1538-7445.AM2014-3574