Mouse Embryonic Fibroblast Cell Culture and Stimulation.

Culture of mouse embryonic fibroblast (MEF) cells represents a powerful system to test gene function due to their easy accessibility, rapid growth rates, and the possibility of a large number of experiments. Fibroblasts are a group of heterogeneous resident cells of mesenchymal origin that have various locations, diverse appearances and distinctive activities. Because of their ubiquitous distribution as tissue cells, these cells are poised to respond to factors released by newly activated innate immune cells, thus becoming a useful tool to study inflammation and immunity.

Measurement of mRNA Decay in Mouse Embryonic Fibroblasts.

mRNA stability control is a critical step in the post-transcriptional regulation of gene expression. Actinomycin D, an antibiotic initially used as an anti-cancer drug, has turned out to be a convenient tool for studying the turnover rates of transcripts in cells, due to its inhibition of mRNA synthesis. Here, we describe a protocol for the measurement of mRNA decay after adding actinomycin D into the medium of stable fibroblast cell lines derived from wild-type and tristetraprolin (TTP)-deficient mouse embryonic fibroblast (MEF) cultures, as well as a protocol for determining the relative transcript abundance using semi-quantitative real time RT-PCR.

Tristetraprolin (TTP) coordinately regulates primary and secondary cellular responses to proinflammatory stimuli.

TTP is an anti-inflammatory protein that acts by binding to AREs in its target mRNAs, such as Tnf mRNA, and promoting their deadenylation and decay. TNF released from inflammatory cells can then stimulate gene expression in tissue cells, such as fibroblasts. To determine whether TTP could affect the decay of TNF-induced transcripts in fibroblasts, we exposed primary embryonic fibroblasts and stable fibroblast cell lines, derived from WT and TTP KO mice, to TNF.

Global analysis of posttranscriptional gene expression in response to sodium arsenite.

Background: Inorganic arsenic species are potent environmental toxins and causes of numerous health problems. Most studies have assumed that arsenic-induced changes in mRNA levels result from effects on gene transcription.

Objectives: We evaluated the prevalence of changes in mRNA stability in response to sodium arsenite in human fibroblasts.

Myeloid-specific tristetraprolin deficiency in mice results in extreme lipopolysaccharide sensitivity in an otherwise minimal phenotype.

Tristetraprolin (TTP) is a mRNA-destabilizing protein that binds to AU-rich elements in labile transcripts, such as the mRNA encoding TNF, and promotes their deadenylation and degradation. TTP-deficient (knockout [KO]) mice exhibit an early-onset, severe inflammatory phenotype, with cachexia, erosive arthritis, left-sided cardiac valvulitis, myeloid hyperplasia, and autoimmunity, which can be prevented by injections of anti-TNF Abs, or interbreeding with TNF receptor-deficient mice. To determine whether the excess TNF that causes the TTP KO phenotype is produced by myeloid cells, we performed myeloid-specific disruption of Zfp36, the gene encoding TTP.

Viperin is required for optimal Th2 responses and T-cell receptor-mediated activation of NF-kappaB and AP-1.

Viperin (virus inhibitory protein, endoplasmic reticulum [ER]-associated, interferon-inducible) has been identified as a highly inducible ER protein that has antiviral activity. Here, we characterized the phenotype of mice deficient in viperin and examined the biological function of viperin in peripheral T-cell activation and differentiation. Splenic CD4(+) T cells deficient in viperin exhibited normal anti-T-cell receptor (TCR)-induced proliferation and IL-2 production, but produced significantly less T helper 2 (Th2) cytokines, including IL-4, IL-5, and IL-13, in association with impaired GATA3 activation, after stimulation with anti-CD3 antibody, which was not restored upon costimulation with anti-CD28.

Coupled induction of iNOS and p53 upregulation in renal resident cells may be linked with apoptotic activity in the pathogenesis of progressive IgA nephropathy.

In situ hybridization, immunohistochemistry, and TUNEL staining were applied to renal biopsy specimens of immunoglobulin A nephropathy (IgAN) patients to determine the expression of nitric oxide synthase (iNOS) (mRNA and protein), p53, and their potential roles in renal cell apoptosis in relation to the development of pathologic lesions. Fifty-one cases were categorized into four subgroups (A-D) according to the presence of progressive histopathological features. A cell type-specific and differential overexpression of iNOS mRNA and protein was demonstrated in glomerular cells in subgroups (A-C) and was found to correlate well with the upregulation of p53 protein by glomerular endothelium and epithelium in early- and advanced-stage disease.

Role of differential and cell type-specific expression of cell cycle regulatory proteins in mediating progressive glomerular injury in human IgA nephropathy.

The activities of cell cycle regulatory proteins have been reported to be associated with the development of pathological lesions in glomerulonephritis. To assess the cellular mechanisms underlying the mesangial cell proliferation and glomerulosclerosis in progressive human IgA nephropathy (IgAN), we examined the expression of E2F1, Rb, c-Myc, proliferating cell nuclear antigen (PCNA), cyclins (D1, E and A), cyclin-dependent kinase 2 (CDK2) and CDK inhibitors (p21(waf1), p27(kip1), 57(kip2) and p16(ink4a)) by immunohistochemistry in renal biopsy specimens. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was also performed to detect the presence of apoptosis.

Downregulation of Bcl-2 by podocytes is associated with progressive glomerular injury and clinical indices of poor renal prognosis in human IgA nephropathy.

Bcl-2 defines a new class of proto-oncogenes that block cell death without promoting cell proliferation. To elucidate the role of Bcl-2 in the development of glomerular lesions in human IgA nephropathy (IgAN), we applied immunohistochemistry coupled with in situ hybridization to detect the expression of Bcl-2 products and their association with Bax, p27(kip1), and p57(kip2) in modulating the apoptotic, proliferative, and sclerotic events in progressive glomerular injury. Glomerular cell apoptosis was examined by TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining.