title: Lipopolysaccharide (LPS) directly suppresses growth hormone receptor (GHR) expression through MyD88-dependent and -independent Toll-like receptor-4/MD2 complex signaling pathways.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Department of Pediatrics, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Prapai
authors.initials: P
authors.lastname: Dejkhamron
authors.forename: Jamuna
authors.initials: J
authors.lastname: Thimmarayappa
authors.forename: Kateryna
authors.initials: K
authors.lastname: Kotlyarevska
authors.forename: Jinhong
authors.initials: J
authors.lastname: Sun
authors.forename: Chunxia
authors.initials: C
authors.lastname: Lu
authors.forename: Erin L
authors.initials: EL
authors.lastname: Bonkowski
authors.forename: Lee A
authors.initials: LA
authors.lastname: Denson
authors.forename: Ram K
authors.initials: RK
authors.lastname: Menon
mesh_terms: D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D046912:Multiprotein Complexes; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011986:Receptors, Somatotropin / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*
chemical_list: D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96; D046912:Multiprotein Complexes; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D011986:Receptors, Somatotropin; D051197:Toll-Like Receptor 4