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. We documented the lack of TTP expression in LPS-stimulated bone marrow-derived macrophages from the mice, whereas fibroblasts expressed TTP mRNA and protein normally in response to serum. The mice exhibited a minimal phenotype, characterized by slight slowing of weight gain late in the first year of life, compared with the early-onset, severe weight loss and inflammation seen in the TTP KO mice. Instead, the myeloid-specific TTP KO mice were highly and abnormally susceptible to a low-dose LPS challenge, with rapid development of typical endotoxemia signs and extensive organ damage, and elevations of serum TNF levels to 110-fold greater than control. We conclude that myeloid-specific TTP deficiency does not phenocopy complete TTP deficiency in C57BL/6 mice under normal laboratory conditions, implying contributions from other cell types to the complete phenotype. However, myeloid cell TTP plays a critical role in protecting mice against LPS-induced septic shock, primarily through its posttranscriptional regulation of TNF mRNA stability.

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. Th2 differentiation of viperin-deficient naive T cells was also impaired in the presence of strong TCR signaling and minimum IL-4, but not under optimal Th2-skewed conditions. In parallel, viperin-deficient T cells showed decreases in NF-kappaB1/p50 and AP-1/JunB DNA binding activities after TCR engagement. Thus, viperin facilitates TCR-mediated GATA-3 activation and optimal Th2 cytokine production by modulating NF-kappaB and AP-1 activities.

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. Here, we describe procedures for mouse embryonic fibroblast cell isolation, primary culture, and stimulation. Specifically, we have optimized a step of serum starvation prior to stimulation. This step is necessary to maintain the quiescent status of these cells before they are exposed to pro-inflammatory stimuli for optimal responses. As shown in our previous studies, these mouse fibroblasts do not express Tnf, Csf2, or Il2 mRNAs at levels readily detectable by routine northern blotting techniques (Lai WS et al., 2006).

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. Northern blotting or NanoString n-Counter are alternative methods to measure mRNA abundance, which is quantified using a phosphorimager in the former case. This protocol is suitable for studying primary cultured cells and stable cell lines derived from transgenic mice and their respective controls, and provides for direct comparisons of mRNA decay rates in otherwise identical cells with and without the gene of interest.

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. METHODS: We used microarray analyses to determine changes in steady-state mRNA levels and mRNA decay rates following 24-hr exposure to noncytotoxic concentrations of sodium arsenite, and we confirmed some of these changes using real-time reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: In arsenite-exposed cells, 186 probe set-identified transcripts were significantly increased and 167 were significantly decreased. When decay rates were analyzed after actinomycin D treatment, only 4,992 (9.1%) of probe set-identified transcripts decayed by > 25% after 4 hr. Of these, 70 were among the 353 whose steady-state levels were altered by arsenite, and of these, only 4 exhibited significantly different decay rates between arsenite and control treatment. Real-time RT-PCR confirmed a major, significant arsenite-induced stabilization of the mRNA encoding δ aminolevulinate synthase 1 (ALAS1), the rate-limiting enzyme in heme biosynthesis. This change presumably accounted for at least part of the 2.7-fold increase in steady-state ALAS1 mRNA levels seen after arsenite treatment. This could reflect decreases in cellular heme caused by the massive induction by arsenite of heme oxygenase mRNA (HMOX1; 68-fold increase), the rate-limiting enzyme in heme catabolism. CONCLUSIONS: We conclude that arsenite modification of mRNA stability is relatively uncommon, but in some instances can result in significant changes in gene expression.

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. The decay rates of transcripts encoded by several early-response genes, including Cxcl1, Cxcl2, Ier3, Ptgs2, and Lif, were significantly slowed in TTP-deficient fibroblasts after TNF stimulation. These changes were associated with TTP-dependent increases in CXCL1, CXCL2, and IER3 protein levels. The TTP-susceptible transcripts contained multiple, conserved, closely spaced, potential TTP binding sites in their 3'-UTRs. WT TTP, but not a nonbinding TTP zinc finger mutant, bound to RNA probes that were based on the mRNA sequences of Cxcl1, Cxcl2, Ptgs2, and Lif. TTP-promoted decay of transcripts encoding chemokines and other proinflammatory mediators is thus a critical post-transcriptional regulatory mechanism in the response of secondary cells, such as fibroblasts, to TNF released from primary immune cells.

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. A total of 51 IgAN cases were categorized into four subgroups (A to D) according to the severity of their histopathological lesions. Creatinine levels, creatinine clearance, and magnitude of proteinuria based on 24-h urine collections at the time of diagnostic renal biopsy were available for the majority of subjects. Bcl-2 expression was observed predominantly in podocytes in IgAN. Podocyte expression of Bcl-2 was found to be upregulated in early-stage disease and downregulated in late-stage disease. Bcl-2 downregulation in progressive IgAN was associated with an increased Bax/Bcl-2 ratio in glomerular epithelial cells and correlated with the downregulation of high endogenous podocyte p27(kip1) and p57(kip2) expression. Bax/Bcl-2 ratios positively correlated with glomerular cell apoptosis and the degree of glomerulosclerosis, whereas p27(kip1) and p57(kip2) expression levels were inversely correlated with mesangial hypercellularity and glomerulosclerosis. Clinicopathologic correlations demonstrated that downregulation of Bcl-2 protein expression was associated with indices of poor renal prognosis in human IgAN. The results suggest that Bcl-2 expression by podocytes may exert modulatory effects on cellular processes that contribute to progressive glomerular injury and play an important role in determining renal outcome in human IgA nephropathy.

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. In the tubulointerstitium, induction of iNOS products was evident in damaged tubules in late-stage disease, in parallel with the upregulation of p53 protein levels in these tubules. Increased TUNEL staining observed in glomeruli with progressive lesions and tubules with degenerative changes positively correlated with the expression levels of iNOS and p53 in glomerular endothelium, epithelium, and their overexpression in damaged tubules. Clinicopathologic correlations demonstrated that induction of iNOS products in renal cells was associated with indices of poor renal prognosis in human IgAN. The coupled induction of iNOS and p53 upregulation in intrinsic renal cells of IgAN may be linked with both pro- and anti-apoptotic activities, thus playing an important role in mediating progressive renal injury and determining renal outcome in human IgAN.

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. In total, 51 cases of IgAN were categorized into four subgroups according to histological severity. A dramatic upregulation of E2F1 expression in mesangial cells was identified in proliferating glomeruli, which correlated well with the proliferation index. High endogenous expression of p27(kip1) and p57(kip2) by podocytes in normal glomeruli and glomeruli with minor lesions was observed to decrease in proliferating and sclerosing glomeruli; this pattern displayed a strong inverse correlation with the mean glomerulosclerosis score and the index of glomerular lesion. Increased apoptotic activity was identified in progressive glomerular lesions of advanced IgAN, which correlated with the proliferative activity in these lesions as assessed by total expression levels of PCNA and CDK2 in glomeruli, E2F1 expression levels in the mesangium, cyclin D1 expression levels in endothelium and the c-Myc glomerular staining score. Our results suggest that the onset and magnitude of mesangial cell proliferation and glomerulosclerosis is associated with the upregulation of E2F1 by mesangial cells and the downregulation of p27(kip1) and p57(kip2) by glomerular epithelial cells. The cell type-specific and coordinated regulation of proliferative and proapoptotic activities of cell cycle regulatory proteins may play an important role in mediating progressive glomerular injury in human IgAN.