The Biotransformation Of Endobiotics By Sulfonation

Child Health And Human Development

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Abstract

The Section on Steroid Regulation investigates molecular mechanisms and biologic implications of modifying substances by sulfonation, a fundamental process in the biotransformation of endobiotics as well as drugs and xenobiotics. Sulfonation, the transfer of an SO3-1 group from the universal donor molecule 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to an acceptor molecule, is essential for normal growth and development as well as maintenance of the internal milieu. Sulfonated macromolecules such as glycosaminoglycans and proteoglycans are involved in cell surface and connective tissue structures and bone formation. Tyrosine sulfonation is a widespread post-translational modification of many secretory and membrane proteins. Glycoprotein hormones are modified by sulfonation of specific saccharide moieties creating unique structural motifs with important functional implications. Low molecular weight compounds such as catecholamines, iodothyronines, neuroendocrine peptides and cholesterol along with its metabolites bile acids, oxysterols, vitamin D and steroid hormones are also modified by sulfonation. By modulating the availability of biologically active hormones, sulfonation can influence biologic activity regardless of whether these compounds act in their unconjugated or sulfoconjugated state or whether they act via a genomic or nongenomic mechanism. Thus, sulfoconjugating enzymes, i.e. sulfotransferases, play an essential role in specific physiologic systems as well as associated disorders. Enzymes sulfoconjugating neutral steroids and sterols comprise a family of sulfotransferases that are members of a super family of cytosolic sulfotransferases (SULT). The steroid/sterol family designated SULT2 is further divided into two subfamilies, i.e. SULT2A1 and SULT2B1. The SULT2A1 subfamily consists of a single form, whereas the SULT2B1 subfamily consists of two isoforms (SULT2B1a and SULT2B1b) that result from of an alternative exon 1 and differential splicing. While SULT2A1 has a broad substrate predilection, the SULT2B1 isoforms have narrower substrate preferences that are confined to selective steroids. For example, SULT2B1a avidly sulfonates pregnenolone, whereas SULT2B1b functions as the physiologic cholesterol sulfotransferase. The SULT2B1 isoforms are differentially expressed, e.g. SULT2B1a is only expressed in the central nervous system where pregnenolone sulfate functions as an important neurosteroid, whereas SULT2B1b is expressed in organs such as skin and retinal tissue. Expression of the gene encoding for the SULT2B1b isozyme in skin is consistent with the involvement of cholesterol sulfate in keratinocyte development. There is a progressive expression of SULT2B1b mRNA, protein and activity in primary cultures of normal human epidermal keratinocytes (NHEK) during calcium-induced differentiation. Notably, the time-course of SULT2B1b expression is similar to that of the nuclear receptor RORalpha. This is significant since cholesterol sulfate is a natural ligand for RORalpha. Furthermore, the expression patterns for SULT2B1b and RORalpha are identical in normal human skin where they are confined to the epidermal granular layer indicating that they are late markers of keratinocyte differentiation. The confinement of cholesterol sulfotransferase to the granular layer of the living epidermis beneath the stratum corneum along with the knowledge that this region of the epidermis contains the highest content of cholesterol sulfate strongly suggests that the principal function of this sulfolipid is carried out primarily in the region of the granular-stratum corneum junction, an action that may, in part, involve the RORalpha nuclear receptor. The epidermis is a perpetually renewing tissue whereby keratinocytes arise from stem cells in the basal layer, move through a series of cellular differentiation events until as dead squames they are finally sloughed off from the outer stratum corneum. We have investigated the expression of SULT2B1b using NHEK as well as immortalized but highly differentiated human keratinocytes (HaCaT cells). The gene for human SULT2B1 contains neither a canonical TATAAA nor a CCAAT motif in the SULT2B1b promoter, nor does an initiator motif overlap the transcription start site (TSS). Many TATA-less promoters are characterized by the presence of multiple GC boxes, which bind the Sp1 transcription activator forming a central role in the assembly of the transcription complex of these promoters, and the gene for SULT2B1 appears to be similarly regulated. That is, the promoter of SULT2B1b contains multiple GC/GT boxes, and mutation of specific Sp1 motifs suggests involvement in transcriptional regulation, a finding supported by deletion analyses. Importantly, nuclear extracts from HaCaT cells contain proteins that bind to probes incorporating Sp1 motifs implicated in gene regulation; furthermore, confirmation of the presence of Sp1 and Sp2 proteins in the HaCaT cell nuclear extracts was obtained by supershift analyses. Notably, a key Sp1 regulatory element is located in the 5?-UTR, and promoter activity within the 5?-UTR is commonly due to the presence of functional Sp1 binding elements. Co-transfection of HaCaT cells with Sp1 and/or Sp2 expression vectors produced dose-dependent increases in promoter activity, although transcriptional activation by Sp2 was less potent than that produced by Sp1. Interestingly, co-expression of Sp1 and Sp2 in suboptimal amounts produced a synergistic effect. Use of a histone deacetylase (HDAC) inhibitor resulted in a dramatic augmentation in reporter gene activity induced by Sp1, an effect not seen with Sp2. It was concluded that, whereas Sp1 functions as a transactivator of the SULT2B1 gene regulating expression of the SULT2B1b isoform, the role of Sp2 is less clear despite its apparent ability to enhance the effect of Sp1 in experiments where HDAC inhibition was not employed. That Sp1 stimulated SULT2B1b promoter activity by inducing histone acetylation was suggested when the effect of HDAC inhibition was lost with mutation of the regulatory Sp1-binding sites. A major hurdle in activating transcription is the presence of a nucleosomal barrier that limits access of the transcription machinery to DNA templates. Relief of nucleosomal repression can be achieved by modification of chromatin components, particularly histone acetylation, which promotes gene transcription by making promoter sequences accessible to transcription factors. Additionally, acetylation of nonhistone proteins, e.g. transcription factors such Sp1 also occurs, which can have regulatory implications. In addition to cholesterol, SULT2B1b will also sulfonate the oxysterol, 7-ketocholesterol (7kCh). The significance of this relates to age-related macular degeneration, a complex disease that involves the aging process as well as genetics and environmental factors. The accumulation of cholesterol in Bruch?s membrane as a process of aging as well as the epidemiologic association to atherosclerosis suggests a mechanistic relation between the two diseases. The accumulation low-density lipoproteins (LDL) in arteries and its subsequent oxidation and ingestion by macrophages is critical in the formation of atherosclerotic plaques. oxLDL is also cytotoxic when added to retinal pigment epithelium (RPE) cells. Oxidation of cholesterol within LDL particles generates a series of cholesterol oxides with potent pharmacological activities, including induction of apoptosis and necrosis. Prolonged oxidation of LDL shows increased cytotoxicity when added to ARPE19 rat retinal cells in culture, and analysis of the oxLDL shows predominance of 7kCh. Furthermore, addition of free 7kCh to retinal ARPE19 cells is markedly cytotoxic; however, SULT2B1b is selectively expressed by these cells, and the sulfoconjugation of 7kCh is protective of the retinal cells.