Expression of the rat liver haptoglobin gene is mediated by isoforms of C/EBPalpha, -beta and -delta proteins

CCAAT/enhancer binding proteins (C/EBP) alpha, -beta and -delta play an important role in mediating I interleukin-6 (IL-6) dependent expression of acute-phase protein (APP) genes in liver during acute-phase (AP) response. Based on the presence of type IL-6 responsive element (IL-6 RE) in the rat haptoglobin (Hp) gene promoter we assumed that some C/EBPalpha, -beta and/or -delta isoforms could mediate the expression of this gene during turpentine-induced AP response. By Western immunoblot and Northern blot assays, we found that turpentine treatment of rats led to a coordinate induction of C/EBPbeta and -delta as well as repression of C/EBPalpha isoforms pool levels in rat liver nuclear extracts (NEs) which was preceded by an adequate alteration of their mRNAs expression in liver. Consequently, results of DNA affinity chromatography revealed that affinity of certain C/EBPalpha isoforms to bind the type I IL-6 RE within the rat Hp gene promoter decreased whereas affinities of certain C/EBPbeta isoforms and C/EBPdelta were increased and induced, respectively. Our data suggest that turpentine-induced alterations of C/EBPalpha, -beta and -delta pool levels and DNA-binding activities can be regarded as an integral part of activation of the Hp gene expression in the course of AP response.     

Structure, hormonal regulation, and identification of the interleukin-6- and dexamethasone-responsive element of the rat haptoglobin gene.

Hepatic expression of the haptoglobin (Hp) gene in mammalian species is stimulated severalfold during an acute-phase reaction. To identify the molecular mechanism responsible for this regulation, the single-copy rat Hp gene has been isolated. The genomic sequences showed a high degree of homology with the primate Hp gene. Activity of the rat Hp gene was increased in cultured liver cells by interleukin-1 (IL-1), IL-6, and glucocorticoids. The genomic Hp gene sequence spanning from -6500 to +6500, when transiently introduced into human hepatoma (HepG2) cells, directed IL-6- and dexamethasone-stimulated expression of rat Hp mRNA and protein. No response to IL-1 was detected, suggesting that the corresponding regulatory element(s) might lie outside of the tested gene sequences. An IL-6- and dexamethasone-responsive element has been localized to the promoter proximal region -146 to -55. Although the nucleotide sequences of this rat Hp gene region showed substantial divergence from that of the human gene, analysis of sequential 5' and 3' deletion constructs indicated an arrangement of functional IL-6 response elements in the rat Hp promoter sequence comparable to that of the human homolog. The magnitude of IL-6 regulation through the rat Hp gene promoter was severalfold lower than that of the human Hp gene. The reduced activity could be ascribed to a single-base difference in an otherwise conserved sequence corresponding to an active element in the human gene. The IL-6 response of the rat Hp element was improved severalfold by substituting that base with the human nucleotide.     

Proximal tubule haptoglobin gene activation is an integral component of the acute kidney injury "stress response"

Haptoglobin (Hp) synthesis occurs almost exclusively in liver, and it is rapidly upregulated in response to stress. Because many of the pathways that initiate hepatic Hp synthesis are also operative during acute kidney injury (AKI), we tested whether AKI activates the renal cortical Hp gene. CD-1 mice were subjected to six diverse AKI models: ischemia-reperfusion, glycerol injection, cisplatin nephrotoxicity, myoglobinuria, endotoxemia, and bilateral ureteral obstruction. Renal cortical Hp gene induction was determined either 4-72 h or 1-3 wk later by measuring Hp mRNA and protein levels. Relative renal vs. hepatic Hp gene induction during endotoxemia was also assessed. Each form of AKI induced striking and sustained Hp mRNA increases, leading to ～10- to 100-fold renal Hp protein elevations (ELISA; Western blot). Immunohistochemistry, and isolated proximal tubule assessments, indicated that the proximal tubule was the dominant (if not only) site of the renal Hp increases. Corresponding urinary and plasma Hp elevations were surrogate markers of this response. Endotoxemia evoked 25-fold greater Hp mRNA increases in kidney vs. liver, indicating marked renal Hp gene reactivity. Clinical relevance of these findings was suggested by observations that urine samples from 16 patients with established AKI had statistically higher (～12×) urinary Hp levels than urine samples from either normal subjects or from 15 patients with chronic kidney disease. These AKI-associated urinary Hp increases mirrored those seen for urinary neutrophil gelatinase-associated lipoprotein, a well accepted AKI biomarker gene. In summary, these studies provide the first evidence that AKI evokes rapid, marked, and sustained induction of the proximal tubule Hp gene. Hp's known antioxidant, as well as its protean pro- and anti-inflammatory, actions imply potentially diverse effects on the evolution of acute tubular injury.     

HMG-1 enhances HMG-I/Y binding to an A/T-rich enhancer element from the pea plastocyanin gene.

High-mobility-group proteins HMG-1 and HMG-I/Y bind at overlapping sites within the A/T-rich enhancer element of the pea plastocyanin gene. Competition binding experiments revealed that HMG-1 enhanced the binding of HMG-I/Y to a 31-bp region (P31) of the enhancer. Circularization assays showed that HMG-1, but not HMG-I/Y, was able to bend a linear 100-bp DNA containing P31 so that the ends could be ligated. HMG-1, but not HMG-I/Y, showed preferential binding to the circular 100-bp DNA compared with the equivalent linear DNA, indicating that alteration of the conformation of the DNA by HMG-1 was not responsible for enhanced binding of HMG-I/Y. Direct interaction of HMG-I/Y and HMG-1 in the absence of DNA was demonstrated by binding of 35S-labeled proteins to immobilized histidine-tagged proteins, and this was due to an interaction of the N-terminal HMG-box-containing region of HMG-1 and the C-terminal AT-hook region of HMG-I/Y. Kinetic analysis using the IAsys biosensor revealed that HMG-1 had an affinity for immobilized HMG-I/Y (Kd = 28 nM) similar to that for immobilized P31 DNA. HMG-1-enhanced binding of HMG-I/Y to the enhancer element appears to be mediated by the formation of an HMG-1-HMG-I/Y complex, which binds to DNA with the rapid loss of HMG-1.