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. Sulfolipids are concentrated in the brain, peripheral nerves and reproductive tissues. 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 importantly 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. Importantly, the SULT2B1 isoforms are differentially expressed. For example, in the mouse, SULT2B1a is exclusively expressed in the central nervous system where pregnenolone sulfate functions as an important neurosteroid. On the other hand, in both human and mouse, SULT2B1b is selectively expressed in skin where cholesterol sulfate plays an important regulatory role. Quantitative expression of the gene encoding for the SULT2B1b isozyme is consistent with the involvement of cholesterol sulfate in keratinocyte development, i.e. there is a progressive expression of SULT2B1b mRNA, protein and activity in primary cultures of normal human epidermal keratinocytes (NHEK) during calcium-induced differentiation. The physiologic import of cholesterol sulfate in the epidermis is in part understood by the knowledge of where during keratinocyte development SULT2B1b is expressed. In this regard, immunocytochemical analysis revealed that expression of SULT2B1b is confined to the granular layer of the epidermis suggesting it is a late marker 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. The epidermis is a perpetually renewing tissue whereby keratinocytes arise from stem cells in the basal layer, move through a seeries of cellular differentiation events until as dead squames they are finally sloughed off from the outer stratum corneum. The fact that ~90% of the cholesterol sulfate formed during calcium-induction of keratinocyte differentiation is found in the cellular membrane fraction with only 10% being present in the soluble cell fraction suggests that this is an important determinant of its physiologic effect. Because of the importance of cholesterol sulfate in the epidermis of human skin we have investigated the expression of SUT2B1b using NHEK as well as immortalized but highly differentiated human keratinocytes (HaCaT) cells. The gene for SULT2B1 contains neither a canonical TATAAA nor a CCAAT motif in the upstream region flanking exon 1B, nor is there an initiator motif. The start of transcription as determined by RLM-RACE yielded multiple transcription start sites (TSS) that tend to cluster in two areas. While TATA-less promoters can direct transcription initiation from multiple sites, our provisional conclusion, based on additional RT-PCR experiments, is that the TSS for SULT2B1b most likely involves a single site located between nt -179 and nt -191 relative to the ATG translation initiator codon. 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. Regulation of the gene for SULT2B1 appears to be similarly under the influence of the Sp1 family of transcription factors. That is, the area upstream of the coding region of SULT2B1b contains multiple GC/GT boxes and mutational analyses suggest involvement of specific motifs in transcriptional regulation. Additionally, deletion analyses correctly confirms the mutational 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. Additional support for the involvement of Sp1 and Sp2 in transcriptional regulation was obtained by co-transfection experiments using NHEK and HaCaT cells. Assuming the location of the TSS for SULT2B1b is correct indicates that a key Sp1 regulatory element is located in the 5?-UTR. Promoter activity within the 5?-UTR is commonly due to the presence of functional Sp1 binding elements. Cholesterol sulfate also has important regulatory roles in human platelets, i.e. it potentiates arachidonic acid-, ADP-, and thrombin-induced platelet aggregation, as well as serotonin secretion. The content of cholesterol sulfate in platelets has been determined to be 566?62 pmol/one billion platelets. That platelet cholesterol sulfate can be produced locally and not just taken up form the circulation has been confirmed by the presence of platelet SULT2B1b. In fact, as in skin, platelet SULT2B1b is the only SULT2 enzyme expressed by these discoid anucleate particles. Although platelets lack a nucleus, they do contain rough endoplasmic reticulum and polysomes and are known to engage in protein synthesis. It is recognized that human platelets and plasma lipoproteins interact and are intimately involved in the pathogenesis of atherosclerosis, thrombosis, and coronary artery disease. Based on real-time PCR, the level of SULT2B1b mRNA in platelets is stably maintained at 4?C but is markedly diminished over a period of 4 h at 37?C. The loss of SULT2B1b mRNA, however, is significantly reduced in the presence of HDL but not LDL. Furthermore, the stabilizing influence of HDL is due specifically to its apolipoprotein A-I component, whereas the apolipoproteins A-II and E are without effect. Importatnly, there is a direct correlation between platelet SULT2B1b mRNA and protein levels that is reflected in enzymatic activity and cholesterol sulfate formation.
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