Charles A. Strott, MD, Head, Section on Steroid Regulation
Young C. Lee, PhD, Staff Scientist
Hirotoshi Fuda, PhD, Research Fellow
Motohiro Endo, MD, PhD, Postdoctoral Fellow
Osamu Hanyu, MD, PhD, Postdoctoral Fellow
Norman B. Javitt, MD, PhD, Adjunct Investigator
Zachary Javitt, Summer Student
We investigate molecular mechanisms and biologic implications of sulfonation, a fundamental process in the biotransformation of endobiotics, drugs, and xenobiotics. Sulfonation, the transfer of an SO3-1 group from the universal sulfonate donor molecule 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to an acceptor compound, is essential for normal growth and development as well as for maintenance of the internal milieu. We have undertaken extensive investigations of sulfotransferases as well as of PAPS synthetases—the bifunctional enzymes that produce the sulfonate donor—to understand their biochemistry, protein structure/function, and molecular biology. Of the two classes of sulfotransfersases, one is tightly associated with membranes, especially the Golgi complex, while the other is composed of a superfamily of soluble or cytosolic enzymes (designated SULT). The former class is responsible for the sulfonation of macromolecules such as glycosaminoglycans and proteoglycans, whereas the SULT class of enzymes is engaged in the sulfonation of low-molecular weight compounds, such as catecholamines, iodothyronines, neuroendocrine peptides, and cholesterol along with its metabolites, oxysterols, bile acids, vitamin D, and steroids. We currently focus on the SULT class of enzymes.
Transcriptional regulation of the human SULT2B1 gene encoding cholesterol sulfotransferase
Lee, Luu,1 Strott; in collaboration with Higashi
As an initial foray into the regulation of the SULT2B1 gene, we examined in detail the more downstream or proximal promoter region. The DNA sequence of the SULT2B1b 5′-flanking region extending from -1 to -548 (relative to ATG) contains several potential transcription factor binding sites, including Ets-1, Ets-2, Pit-1a, NF-1, AP-1, AP-2, and AP-4. When we mutated these sites individually and examined them by transfection, we noted either no effect on promoter activity or only modest decreases. In addition, we identified five Sp1 sites, of which two demonstrated regulatory activity: one regulatory Sp1 site is upstream of the start of transcription while the other regulatory Sp1 site is located within the 5′UTR of SULT2B1b mRNA. We obtained support for the notion that the Sp1 family regulates the human SULT2B1 gene when we found that nuclear extracts from HaCaT (immortalized human keratinocytes) cells expressing SULT2B1b contain proteins that bind to probes bearing regulatory Sp1 sites; we also found Sp1 and Sp2 proteins in the nuclear extracts. Furthermore, 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; however, co-expression of Sp1 and Sp2 in suboptimal amounts produced a synergistic effect. Histone deacetylase inhibition caused a dramatic augmentation in reporter gene activity induced by Sp1, an effect not seen with Sp2. We concluded that Sp1 clearly functions as a transactivator of the SULT2B1 gene by regulating expression of the SULT2B1b isoform, whereas the role of Sp2 is less clear despite its apparent ability to enhance the effect of Sp1.
Regulation of the human SULT2B1 gene by DNA methylation
Lee, Luu, 1 Chan, 2 Strott; in collaboration with Higashi
While SULT2B1b is selectively expressed in a tissue-specific manner, e.g., in skin, placenta, and prostate, the SULT2B1a isoform is essentially globally silenced. DNA analysis revealed that the proximal promoter regions of genes encoding both SULT2B1 isoforms contain several CpG dinucleotides in which the cytosines are subject to methylation. Indeed, the SULT2B1a promoter is hypermethylated in three human cell types—normal human epidermal keratinocytes (NHEK), immortalized human keratinocytes (HaCaT), and human embryonic kidney 293T cells—that do not express the isoform. In addition, the SULT2B1b promoter in 293T cells, which do not express the isoform, is similarly hypermethylated. In contrast, the proximal promoter of SULT2B1b in NHEK, which highly express the isoform, is completely unmethylated. In the case of hypermethylated promoters, removal of the methyl groups leads to a striking induction of expression. Conversely, in vitro methylation of SULT2B1a and SULT2B1b promoter/reporter constructs markedly reduces promoter activity after transfection into NHEK. It thus seems clear that expression of the SULT2B1 isoforms is regulated, at least in part, by methylation of CpG dinucleotides in their proximal promoter regions. This finding suggests an explanation for both the global silencing of SULT2B1a and the tissue-specific expression of SULT2B1b. Methyl-CpG-binding proteins use transcriptional co-repressor molecules to silence transcription and modify surrounding chromatin, thus providing a link between DNA methylation and chromatin remodeling. Preliminary ChIP analysis indicates that methylated DNA-binding proteins as well as DNA methyltransferases and histone deacetylases are recruited to the region of the proximal promoter of SULT2B1a that is rich in methylated CpG dinucleotides.
Cloning and characterization of the rat SULT2B1 gene
Kohjitani, 3 Fuda, Hanyu, Strott
The SULT2B1 gene is unique among the steroid/sterol sulfotransferase genes in that it encodes two isoforms as a result of an alternative exon I. The two isoforms are thus identical except for unique amino-terminal ends. Interestingly, whereas the orthologous human and mouse SULT2B1 gene structures are identical, the rat SULT2B1 gene structure diverges. Similar to human and mouse SULT2B1 genes, the rat SULT2B1 gene consists of an alternative exon I; however, as a result of exonic rearrangement, the genic locations of exons IA and IB are reversed in the rat gene. Whereas exon IA is located downstream of exon IB in human and mouse SULT2B1 genes, exon IA is located upstream of exon IB in the rat SULT2B1 gene. Furthermore, unlike the case of human and mouse SULT2B1 genes, which require differential splicing because a portion of exon IA is fused to exon IB to complete the SULT2B1b mRNA, such splicing is not required with the rat gene. The rearrangement of the rat SULT2B1 gene is particularly noteworthy. Exon IA is not simply relocated upstream of exon IB, which is the reverse of the situation in human and mouse genes, but that portion of exon IA encoding the unique amino terminus of the SULT2B1a isoform is also relocated. If not for this rearrangement, the SULT2B1b protein would sustain a substantial amino acid deletion, which presumably would render it inactive (e.g., it would lack the critical PSB loop). The part of exon IA encoding the common amino acid sequence of the two isoforms remains in the same relative gene position as in the human and mouse genes and becomes exon II in the rat gene. As a result, differential splicing, which is necessary to produce the SULT2B1b mRNA in the human and mouse species, is no longer required in the rat. The mechanism behind this precise exonic cutting and rearrangement is not yet understood.
Regulation of SULT2B1a (pregnenolone sulfotransferase) expression in rat glioma cells
Kohjitani, 3 Fuda, Hanyu, Strott
The neurosteroid pregnenolone sulfate (PS) is synthesized in glial cells and plays significant roles in the regulation of learning and memory performance. We aimed to use the C6 glioma cell line to elucidate the mechanism of steroid/sterol sulfotransferases (SULT) mRNA expression; SULT catalyzes the conversion of pregnenolone to PS. C6 cells express the SULT2B1a isoform but neither the SULT2A1 nor SULT2B1b isoform. Increasing the concentration of L-glutamic acid in the presence of cyclothiazide, which prevents AMPA receptor desensitization, attenuated SULT2B1a mRNA expression, whereas neither NMDA nor kainic acid had a significant effect. Exposure to the synthetic glutamate analogue alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in the presence of cyclothiazide inhibited SULT2B1a expression. The selective AMPA/kainate receptor antagonist 2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) completely reversed, and the specific neuronal nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI) partially reversed, inhibition of SULT2B1a expression by L-glutamic acid. Induction of inducible NOS by combining lipopolysaccharide (LPS) and TNF-alpha dramatically attenuated SULT2B1a expression, which was partially reversed by the specific inducible NOS inhibitor N6-(1-iminoethyl)-L-lysine hydrochloride (L-NIL). Exogenous NO donors inhibited SULT2B1a mRNA expression. Exposure to sodium nitroprusside, LPS/TNF-alpha, and L-glutamic acid with cyclothiazide augmented the production of nitrite, a stable degradation product of NO. These findings suggest that the enzyme SULT2B1a is responsible for the synthesis of PS and that activation of the excitatory amino acid receptors of the AMPA subtype inhibits SULT2B1a mRNA expression by facilitating intracellular NO signaling.
Cholesterol sulfate induction of filaggrin expression: role of the orphan nuclear receptor RORalpha
Hanyu, Fuda, Endo, Strott; in collaboration with Higashi
Recent crystallographic data showed that cholesterol sulfate binds with high affinity to the retinoid-related orphan nuclear receptor (ROR)alpha. Moreover, the high-affinity binding of cholesterol sulfate was found to translate into increased transcriptional activity of ROR. In the mouse, RORalpha mRNA is widely expressed, although RORalpha protein is expressed in the periphery at detectable levels only in skin and testis. Based on its expression in mouse skin, it has been assumed that RORalpha is expressed in human skin although such expression has not been clearly demonstrated. The observation that the granular layer of the human epidermis contains the highest epidermal concentration of cholesterol sulfate, coupled with the observation that SULT2B1b and the barrier protein filaggrin co-localize in the granular layer, suggests the possibility of an important functional association. Further supporting this association, the addition of cholesterol sulfate to culture medium bathing NHEK induced the expression of filaggrin in a dose-dependent fashion. Moreover, cholesterol sulfate binds to the orphan nuclear receptor RORalpha with high affinity, with the latter expressed in the epidermis in a pattern similar to that of SULT2B1b and filaggrin.
As we previously reported for SULT2B1b (cholesterol sulfotransferase) and filaggrin, RORalpha localizes to the outer granular layer of the human epidermis, the first demonstration of the expression and localization of RORalpha in human skin. Primary cultures of basal cells derived from the human epidermis (referred to as normal human epidermal keratinocytes or NHEK) undergo terminal differentiation when subjected to an increase in the calcium concentration of the medium. Under these conditions, SULT2B1b, filaggrin, and RORalpha are induced in a similar manner and time frame; furthermore, of the four subtypes of RORalpha(1-4) based on differences in their amino termini, only subtype 4 is expressed by NHEK. The association of RORalpha with filaggrin expression was clearly demonstrated when siRNA inhibited expression of the gene for RORalpha by NHEK by about 95 percent, resulting in a parallel reduction in the expression of filaggrin by about 80 percent; furthermore, adding cholesterol sulfate to the medium failed to cause a recovery in the expression of filaggrin. Such a result would be expected if cholesterol sulfate acts on filaggrin expression by binding to the RORalpha nuclear receptor. Knocking down the gene for SULT2B1b also led to a reduction in filaggrin expression; in this case, however, the addition of cholesterol sulfate to the medium successfully restored filaggrin expression. Our studies strongly suggest that cholesterol sulfate produced by the SULT2B1b steroid/sterol sulfotransferase activates the gene for filaggrin via its interaction with the orphan nuclear receptor RORalpha. Our work is the first demonstration of a molecular action for cholesterol sulfate that is reminiscent of a typical hormone.
Role of SULT2B1b in protecting against age-related macular degeneration
Javitt N, Fuda, Strott; in collaboration with Rodriquez
Understanding the pathogenesis of age-related macular degeneration, the leading cause of blindness in the elderly, is an important undertaking. This complex disease involves the aging process as well as genetics and environmental factors. The accumulation of cholesterol in Bruch's membrane as a process of aging and the epidemiologic association of cholesterol with atherosclerosis suggest a mechanistic relation between macular degeneration and atherosclerosis. In the latter disease, the accumulation of low-density lipoproteins (LDLs) in arteries and their subsequent oxidation and ingestion by macrophages are believed to be critical in the formation of atherosclerotic plaques. The internalization of oxidized LDL (oxLDL) by macrophages leads to foam cell formation, a process thought to be one of the principal causes of atherosclerosis. The cytotoxicity of oxLDL has also been reported in aortic endothelial cells and retinal pigment epithelium (RPE) cells. The oxidation of esterified and unesterified cholesterol within the LDL particle generates a series of cholesterol oxides known as oxysterols, which have potent pharmacological activities, including induction of apoptosis and necrosis. Using ARPE19 rat retinal cells in culture, oxLDL shows increased cytotoxicity with prolonged oxidation. Analysis of the oxLDL shows predominance of 7-oxygenerated products, including 7alpha-/7beta-hydroxycholesterol and 7-ketocholesterol (7kCh). The addition of these oxysterols to ARPE19 cells in free form indicated that 7kCH is the most cytotoxic. Prolonged oxidation of LDL increases the levels of 7kCh, which appears to account for most of the cytotoxicity associated with oxLDL internalization in ARPE19 cells. Interestingly, SULT2B1b, but not SULT2B1a or SULT2A1, is expressed in retinal ARPE19 cells as well as in the normal monkey retina, and, importantly, SULT2B1b actively sulfonates 7kCh. Furthermore, while free 7kCh is cytotoxic when added to cultures of ARPE19 cells, the sulfoconjugation of 7kCh protects cells from the adverse effects of unesterified 7kCh.
Sterol 27-hydroxylase in the monkey retina
Lee, Fuda, Javitt N, Strott; in collaboration with Rodriquez
Sterol 27-hydroxylase (CYP27A1) is a mitochondrial P-450 enzyme with broad substrate specificity for C27 sterols, including 7kCh. CYP27A1 is widely expressed in human tissues but has not been previously demonstrated in the retina. We examined the expression and localization of CYP27A1 in the monkey retina, where it localized mainly to the photoreceptor inner segments. In addition, we observed CYP27A1 in Müller cells, with faint immunostaining detected in the retinal pigmented epithelium (RPE) and choriocapillaris. We also determined that the 27-hydroxylation of 7kCh rendered it nontoxic to cultured RPE cells. Moreover, when mixed with 7-kCh, 27OH7kCh significantly reduced the toxicity of 7-kCh. Considered in the context of the known functions of CYP27A1, the data imply that its expression in the retina serves to modify the biological activity of oxidized sterols that are either transported or generated locally by photo-oxidation. The strong presence of CYP27A1 in the photoreceptor inner segment hints at an oxysterol neutralization function. Hydroxylation of toxic oxysterols such as 7kCh may be necessary in the oxidative environment of the photoreceptors. Significant in vitro evidence demonstrates photo-oxidation of cholesterol, although the presence of 7kCh and other oxysterols in the retina has not been demonstrated. The attenuation of 7kCh cytotoxicity by 27OH7kCh has not been previously reported, but co-incubation of 7kCh with other oxysterols has shown a protective effect. We suspect that the attenuation effect is attributable to the competition of oxysterols for NADPH oxidase.
Novel role of SULT2B1b in oxysterol metabolism
Fuda, Javitt N, Strott; in collaboration with Ikegawa
Oxysterols constitute a class of cholesterol metabolites that exhibit a broad range of biological effects ranging from cytotoxicity to regulation of nuclear receptors. The role of oxysterols such 7-kCh in the development of retinal macular degeneration and atheromatous lesions is of particular interest, but little is known of metabolic routes for their disposal. We have established that the sterol sulfotransferase, SULT2B1b, which is known to sulfonate cholesterol efficiently, is also capable of effectively sulfonating a variety of oxysterols, including 7-kCh. Using transfection of 293T cells, which do not normally express SULT2B1b, we determined that the level of SULT2B1b expression is associated with a reduction in the toxicity level of 7-kCh. That is, protection from 7-kCh-induced loss of cell viability with transfected 293T cells correlated with an increase in the synthesis of SULT2B1b protein as well as with an increase in the production of 7-kCh sulfate. Furthermore, we found that 7-kCh sulfate, when added to the culture medium of 293T cells in amounts equimolar to 7-kCh, did not cause any loss of cell viability. In addition, we extended the range of C27 sterols that are substrates for SULT2B1b to include 7a- and 7b-hydroxycholesterol as well as the 7-hydroperoxide derivative of cholesterol, thus expanding the potential role of this novel pathway in modulating the injurious effects of both oxysterols and hydroperoxides in vivo.
1 Dai Chu N. Luu, BS, former Postbaccalaureate Fellow, now at Virginia Commonwealth University School of Medicine, Richmond, VA
2 Edward S. Chan, former Summer Student, now at Northwestern University, Feinberg School of Medicine, Chicago, IL
3 Atsushi Kohjitani, DDS, PhD, former Postdoctoral Fellow, now at Okayama University Hospital of Medicine and Dentistry, Okayama, Japan
COLLABORATORS
Yuko Higashi, MD, PhD, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
Shigeo Ikegawa, PhD, Kinki University, Osaka, Japan
Ignacio R. Rodriguez, MD, PhD, Laboratory of Retinal Cell and Molecular Biology, NEI, Bethesda, MD
For further information, contact chastro@mail.nih.gov.
