Esterase 1 is a novel transcriptional repressor of growth hormone receptor gene expression: a unique noncatalytic role for a carboxyesterase protein.

The pleiotropic actions of GH result from its engagement with the GH receptor (GHR). GHR expression is regulated by free fatty acids (FFA). A cDNA phage expression library was screened to identify a phage clone expressing esterase 1 (ES1) binding to the FFA-response element (FARE), L2-D1, in the murine GHR promoter. Ectopically expressed ES1 inhibited GHR promoter activity via effects at two FARE, L2-D1 and L2-A2. Chromatin immunoprecipitation experiments demonstrated specific association of ES1 with the FARE. Catalytically inactive ES1 retained inhibitory activity on the GHR promoter and excluded the possibility that the effect on the GHR promoter was an indirect effect secondary to ES1's actions on the intracellular metabolism of FFA. Ectopically expressed ES1 inhibited the endogenous GHR mRNA and protein expression in 3T3-F442A preadipocytes. Subcellular fractionation and confocal microscopy established that ES1 localizes both to the cytoplasm and the nucleus. Experiments demonstrated chromosome region maintenance 1-dependent nuclear export and the presence of a functional nuclear export signal in ES1. The domain of ES1 responsible for the effect on the GHR promoter was localized to the C-terminal portion of the protein. The in vivo significance of ES1's effect on GHR expression was suggested by decreased liver GHR mRNA expression in mice on a high-fat diet correlating with increased steady-state abundance of liver ES1 mRNA. Our results identify and characterize ES1 as a novel transcriptional regulator of GHR gene expression, thereby establishing a unique nonenzymatic role for a carboxyesterase and expanding the potential biological roles of this protein superfamily.

Lipopolysaccharide (LPS) directly suppresses growth hormone receptor (GHR) expression through MyD88-dependent and -independent Toll-like receptor-4/MD2 complex signaling pathways.

INTRODUCTION: Sepsis is associated with growth hormone (GH) insensitivity and in the intact animal the major surface component of the bacterial cell wall, lipopolysaccharide (LPS), inhibits GH receptor (GHR) gene expression. The prevailing explanation for LPS-induced effects on the GHR promoter is that this effect is indirect via generation of cytokines. Our recent studies demonstrate that saturated free fatty acids (FFAs) inhibit the activity of the murine GHR promoter. Saturated FFAs are an essential component of the lipid A moiety of LPS required for biological activity of LPS. HYPOTHESIS: LPS directly modulates the activity of the dominant GHR promoter via interaction with Toll-like receptor(s) (TLR)/MD2 complex and activation of cognate signaling pathway(s). RESULTS: In transient transfection experiments with RAW 264.7 cells which express endogenous TLR4 and MD2, LPS treatment inhibited GHR promoter activity. Co-transfection of dominant negative TLR4 abrogated this effect on GHR promoter activity. In HEK 293T cells, which are devoid of endogenous TLR4 or MD2, ectopic expression of TLR4 and MD2 resulted in LPS-induced inhibition of the GHR promoter activity. The inhibition of GHR promoter activity was demonstrable by 5-6h after exposure to LPS and persisted at 24h. Fatty-acid free LPS failed to elicit a similar effect on the GHR promoter and the effect of LPS was abrogated by Polymyxin B. The essential role of the cofactor MD2 on the effect of LPS on the GHR promoter was established in experiments using ectopic expression of wild type and mutant MD2. Cotransfection of CD14 in these cells failed to alter the effect of LPS on the activity of the GHR promoter. Analysis of cell culture supernatant excluded the possibility that the effect of LPS was secondary to release of cytokines from the transfected cells. The effect of LPS on the endogenous GHR promoter activity and protein expression was confirmed in F442A preadipocyte cells. In HEK 293T cells, ectopic expression of mutant MyD88 or mutant TRIF abrogated the effect of LPS on the GHR promoter, suggesting that the effect of LPS on the GHR promoter was via both MyD88-dependent and -independent pathways. CONCLUSIONS: LPS acts through both MyD88-dependent and -independent TLR4 signaling pathways to directly inhibit GHR gene expression. Our results establish a novel cytokine-independent mechanism for decrease in GHR expression in bacterial sepsis.

Inhibition of growth hormone receptor gene expression by saturated fatty acids: role of Kruppel-like zinc finger factor, ZBP-89.

The expression and function of the GH receptor is critical for the actions of pituitary GH in the intact animal. The role of systemic factors in the reduced expression of the GH receptor and consequent GH insensitivity in pathological states such as sepsis, malnutrition, and poorly controlled diabetes mellitus is unclear. In the current study, we demonstrate that saturated (palmitic and myristic; 50 microM) fatty acids (FA) inhibit activity of the promoter of the major (L2) transcript of the GH receptor gene; unsaturated (oleic and linoleic) FA (200 microM) do not alter activity of the promoter. Comparable effects with palmitic acid and the nonmetabolizable analog bromo-palmitic acid, and failure of triacsin C to abrogate palmitic acids effects on GH receptor expression indicate that this effect is due to direct action(s) of FA. Palmitic acid, but not the unsaturated FA linoleic acid, decreased steady-state levels of endogenous L2 mRNA and GHR protein in 3T3-L1 preadipocytes. The effect of FA was localized to two cis elements located approximately 600 bp apart on the L2 promoter. EMSA and chromatin immunoprecipitation assays established that both these cis elements bind the Krüppel-type zinc finger transcription factor, ZBP-89. Ectopic expression of ZBP-89 amplified the inhibitory effect of FA on L2 promoter activity and on steady-state levels of endogenous L2 mRNA in 3T3-L1 preadipocytes. Mutational analyses of the two ZBP-89 binding sites revealed that both the sites are essential for palmitic acid's inhibitory effect on the L2 promoter and for the enhancing effect of ZBP-89 on palmitic acid-induced inhibition of the L2 promoter. Our results establish a molecular basis for FA-induced inhibition of GH receptor gene expression in the pathogenesis of acquired GH insensitivity in pathological states such as poorly controlled diabetes mellitus and small for gestational age.