TNF-alpha downregulates murine hepatic growth hormone receptor expression by inhibiting Sp1 and Sp3 binding.

Children with chronic inflammatory diseases experience growth failure and wasting. This may be due to growth hormone resistance caused by cytokine-induced suppression of growth hormone receptor (GHR) gene expression. However, the factors governing inflammatory regulation of GHR are not known. We have reported that Sp1 and Sp3 regulate hepatic GHR expression. We hypothesized that TNF-alpha suppresses GHR expression by inhibiting Sp1/Sp3 transactivators. LPS administration significantly reduced murine hepatic GHR expression, as well as Sp1 and Sp3 binding to GHR promoter cis elements. TNF-alpha was integral to this response, as LPS did not affect hepatic Sp1/Sp3 binding or GHR expression in TNF receptor 1-deficient mice. TNF-alpha treatment of BNL CL.2 mouse liver cells reduced Sp1 and Sp3 binding to a GHR promoter cis element and downregulated activity of a GHR promoter-driven luciferase reporter. Combined mutations within adjacent Sp elements eliminated GHR promoter suppression by TNF-alpha without affecting overall nuclear levels of Sp1 or Sp3 proteins. These studies demonstrate that murine GHR transcription is downregulated by LPS, primarily via TNF-alpha-dependent signaling. Evidence suggests that inhibition of Sp transactivator binding is involved. Further investigation of these mechanisms may identify novel strategies for preventing inflammatory suppression of growth.

Recruitment of a repressosome complex at the growth hormone receptor promoter and its potential role in diabetic nephropathy.

The growth hormone (GH)-GH receptor (GHR) axis modulates growth and metabolism and contributes to complications of diabetes mellitus. We analyzed the promoter region of the dominant transcript (L2) of the murine GHR to determine that a cis element, L2C1, interacts with transcription factors NF-Y, BTEB1, and HMG-Y/I. These proteins individually repress GHR expression and together form a repressosome complex in conjunction with mSin3b. The histone deacetylase inhibitor trichostatin A increases expression of the murine GHR gene, enhances association of acetyl-H3 at L2C1, inhibits formation of the repressosome complex, and decreases NF-Y's association with L2C1. Our studies reveal that murine models of experimental diabetes mellitus are characterized by reduced hepatic GHR expression, decreased acetyl-H3 associated with L2C1, and increased formation of the repressosome complex. In contrast, in the kidney diabetes mellitus is associated with enhanced GHR expression and lack of alteration in the assembly of the repressosome complex, thus permitting exposure of kidneys to the effects of elevated levels of GH in diabetes mellitus. Our findings define a higher-order repressosome complex whose formation correlates with the acetylation status of chromatin histone proteins. The delineation of the role of this repressosome complex in regulating tissue-specific expression of GHR in diabetes mellitus provides a molecular model for the role of GH in the genesis of certain microvascular complications of diabetes mellitus.

Identification and characterization of a novel transcript of the murine growth hormone receptor gene exhibiting development- and tissue-specific expression.

The growth hormone (GH) receptor gene is characterized by heterogeneity in the 5'-untranslated region (UTR). The technique of 5'-rapid amplification of cDNA ends (RACE) was employed to identify potentially novel 5'-UTRs for the GH receptor gene. One of the RACE clones displayed sequence homology to the human V5-UTR; hence this transcript was designated as L5. Sequence analysis of genomic DNA established that L5 was immediately upstream of exon 2. Northern blot analysis indicated that two bands of sizes congruent with4.8 kb, corresponding to GH receptor mRNA, and congruent with1.5 kb corresponding to GH binding protein mRNA, were detectable in liver, skeletal muscle, kidney and heart but not in brain, spleen, lung or testis. Fluorescent 5'-nuclease real-time RT-PCR based analysis indicated that in the placenta and fetal liver, the L5 transcript represented 10-15% of the GH receptor transcripts. In the adult liver, heart and kidney, the L5 transcript is less abundant accounting for 1-5% of the total GH receptor transcripts. Primer extension and ribonuclease protection assays were performed to identify the major transcription start site at 778 bp from the ATG codon. Transient transfection experiments revealed that the 5'-flanking sequence had promoter activity in rat placental trophoblast (HRP.1), Chinese hamster ovary (CHO) and mouse liver (BNL CL.2) cells. Analysis of expression of the L5 transcript in the non-obese diabetic (NOD) mouse, a model of spontaneous autoimmune diabetes, indicated that the expression of the L5 transcript was decreased in liver and kidney by 80-90 and 40-50%, respectively, but expression remained unchanged in the heart.