Arturo Diaz

Regulation of transforming growth factor-beta 1 gene expression by glucocorticoids in normal human T lymphocytes.

Glucocorticoids (GC) modulate immune function in a number of ways, including suppression of T cell proliferation and other IL-2-mediated T cell functions. These inhibitory effects are similar to those induced by transforming growth factor-beta 1 (TGF-beta 1), a cytokine with potent T cell inhibiting activities. We examined the hypothesis that GC effects may be at least partially achieved through modulation of the expression of the TGF-beta 1 gene in activated T cells. Normal T cells were cultured with or without purified phytohemagglutinin (PHA-p) and 4 beta-phorbol 12-myristate 13-acetate (PMA) in the presence or absence of the synthetic GC, dexamethasone (100-200 micrograms/ml). The production of latent and active forms of TGF beta by these cells were analyzed by immunoblotting and bioassays. The steady-state levels of TGF-beta 1 mRNA were analyzed in total RNA from these cells by Northern hybridizations using a human TGF-beta 1 cDNA. The results showed that dexamethasone caused an increase in TGF beta production and a dose-dependent two to fourfold increase in TGF-beta 1 mRNA in activated as well as in unstimulated T cells, 1 h after exposure of the cultures to the steroid. The increase in TGF-beta 1 mRNA levels by dexamethasone was further potentiated two to threefold by cycloheximide, suggesting that the steroid effect may be due to inhibition of the synthesis of proteins that decrease TGF-beta 1 gene transcription or the stability of its transcripts. Finally, in vitro nuclear transcription studies indicated the dexamethasone effects on TGF-beta 1 gene expression to be largely transcriptional.

Interferon-gamma regulates collagen and fibronectin gene expression by transcriptional and post-transcriptional mechanisms

Interferon-gamma (IFN-gamma) regulates the expression of collagen and fibronectin genes by molecular mechanisms not completely understood. We investigated the effects of IFN-gamma on the expression of the genes encoding alpha 1 (I) procollagen (COL1A1), alpha 1 (III) procollagen (COL3A1), and fibronectin (FN) in cultured normal human lung fibroblasts. Labeled newly synthesized proteins were analysed by electrophoresis, mRNA levels and stability by Northern hybridizations, and transcription rates by in vitro assays. IFN-gamma caused a reduction in the production of alpha 1 (I) and alpha 1 (III) procollagens and of fibronectin. The reduction in the production of procollagen chains was shown to result from a combination of IFN-gamma-induced inhibition of the transcription rates of the COL1A1 and COL3A1 genes and destabilization of the corresponding transcripts. IFN-gamma increased the transcription rate of FN, but also decreased the stability of the corresponding transcripts. The net results indicate that the regulation of the expression of extracellular matrix genes by IFN-gamma is a complex process that involves changes in gene transcription rates, alterations in mRNAs stability, and possibly, modulation of the rates of translation.

Effects of interferon-γ and tumor necrosis factor α on the expression of the genes encoding aggrecan, biglycan, and decorin core proteins in cultured human chondrocytes

OBJECTIVE: To determine the effects of interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha), alone or in combination, on the expression of aggrecan, biglycan, and decorin core protein genes in human chondrocytes. METHODS: Isolated human chondrocytes were cultured on poly(2-hydroxyethyl methacrylate)-coated plastic dishes to prevent the loss of cartilage-specific phenotype, and the effects of IFN gamma and TNF alpha, alone or in combination, on aggrecan, biglycan, and decorin core protein gene transcription and steady-state messenger RNA (mRNA) levels were examined. RESULTS: The addition of IFN gamma (1.5 pM) or TNF alpha (0.3 pM) caused a decrease in the steady-state level of aggrecan mRNA (-25% and -15%, respectively), and the combination of these low-concentration cytokines caused a potent inhibition (-66%). These effects were the result of a decrease (-50%) in the transcription rate of the corresponding gene. At the concentrations used, IFN gamma did not alter the levels of biglycan mRNA or the transcription rates of the biglycan core protein gene. In contrast, TNF alpha decreased biglycan steady-state mRNA levels (-62%) and the biglycan core protein gene transcription rate (-18%). The combination of IFN gamma and TNF alpha resulted in a potentiation of the inhibitory effects of TNF alpha on biglycan mRNA levels (-79%) and transcription rate of the biglycan core protein gene (-46%). IFN gamma produced a modest decrease in decorin mRNA levels (-23%) and decorin core protein gene transcription rate (-17%). In contrast, TNF alpha resulted in a marked increase in decorin mRNA levels (+260%) that was not the result of transcriptional regulation. Notably, the combination of IFN gamma and TNF alpha potentiated the inhibitory effects of IFN gamma on decorin mRNA (-80%) and on the transcription of the corresponding gene (-43%). Similar results were obtained in fetal and adult articular chondrocytes. CONCLUSION: These data demonstrate that 1) the expression of the core protein genes encoding the cartilage proteoglycans aggrecan, biglycan, and decorin is differentially regulated by IFN gamma and TNF alpha; 2) these effects are mediated by transcriptional and posttranscriptional mechanisms; and 3) the combination of the 2 cytokines causes a potent inhibitory effect on the expression of the genes for the core proteins of these 3 proteoglycans, which occurs largely at the transcriptional level. The inhibition of aggrecan, decorin, and biglycan core protein gene expression by the combination of IFN gamma and TNF alpha may contribute to the cartilage destruction that is characteristic of inflammatory joint diseases.

Glucocorticoid-mediated inhibition of interleukin-2 receptor α and -β subunit expression by human T cells

To determine the mechanism of glucocorticoid (GC)-mediated inhibition of T cell functions, the effect of dexamethasone (DM) on T cell proliferation and interleukin-2 receptor (IL-2R) generation were studied. Dexamethasone inhibited IL-2-induced T cell proliferation by 30%–88%, relative to its concentration within the cultures. The effect of DM on expression of IL-2Rα (Tac, p55, CD25) and β (p75) genes in activated T cells was examined next. In T cells stimulated with purified phytohemagglutinin (PHA-p) and 4s-phorbol 12-myristate 13-myristate 13-acetate (PMA) addition of DM to the cultures resulted in a 60% reduction in IL-2Rα and a 30% reduction in IL-2Rs membrane expression compared to T cells cultured in the absence of DM (p < 0.01). Inhibition of membrane IL-2Rα and IL-2Rβ expression by 10−6M DM was partially reversible by recombinant human IL-2 (rhIL-2). By Northern blot analysis, DM caused a comparable decrease in IL-2Rα and in IL-2Rs mRNA levels to membrane receptor expression in mitogen-stimulated T cells. By in vitro transcription assays, DM regulated IL-2Rα gene expression at a transcriptional level while transcription of IL-2Rs gene was unaffected by DM. The mechanism of action of DM on IL-2Rα transcription was examined by determining the mRNA levels of the p50 subunit of nuclear factor kappa B (NF-κB), a transcription factor that stimulates IL-2Rα gene expression. The data indicate that 10−6M DM increased T cell p50 NF-κB mRNA levels by four-fold compared to the levels obtained in the absence of DM. Further, the level of nuclear proteins capable of binding to the NF-κB sites in activated T cells increased in response to DM. In sum, DM regulates T cell membrane expression of IL-2R by more than one molecular mechanism.