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RECEPTORS AND SIGNALING, unit from involves the hydroxylation of C-20 INTRACELLULAR RECEPTORS — and C-22 followed by cleavage by desmolase to produce AND NO (C21). Conversion of pregnenolone to pro- gesterone requires oxidation of the 3-hydroxyl group to SCOTT L. DIAMOND a 3-keto group and isomerization of the C-5-C-6 dou- Institute for Medicine and Engineering ble bond to the C-4-C-5 position. Progesterone can be Philadelphia, Pennsylvania metabolized to the major , cortisol (by mul- tiple hydroxylations), or to the major , aldosterone (by multiple hydroxylations and oxidation of OUTLINE the C-18 methyl group to an aldehyde). Alternatively, progesterone can be modified to androgens and Introduction via specific hydroxylation, chain cleavage, and reduction and their reactions. hormones, , and D are Steroid Receptors and Regulation not derived from cholesterol and are unrelated in struc- ture to steroid hormones, but also diffuse across the cell and membrane to bind their intracellular in target Nitric Oxide: cGMP-Dependent Signaling tissues. Nitric Oxide: cGMP-Independent Signaling Nitric Oxide, Peroxynitrite, and Nitrotyrosine STEROID RECEPTORS AND GENE REGULATION between Nitric Oxide and Steroid Signaling Summary: Relevance to Biotechnology Steroid regulation of gene expression involves steroid Acknowledgments crossing the plasmalemma via Bibliography (Fig. 1). Once in the , the steroid binds its receptor with high affinity (Kd ~ 1 nM), which causes the release of chaperones [including heat shock hsp70 INTRODUCTION and hsp90, p23, and various peptidylprolyl isomerase (PPIase)-active immunophilins/cyclophilins (CyP) such as Soluble extracellular molecules that cross the cell FKBP52 (hsp56), CyP18, and CyP40] from the receptor, membrane, bind an , and alter and consequent exposure of nuclear localization sequences cellular are essential regulators of mammalian and sites in the steroid receptor (1,2). The endocrine . This article reviews two of the most chaperones maintain the receptor in a conformation that well-studied and prototypical intracellular receptors: is competent for steroid binding but lacks DNA regulatory steroid receptors, which are transcriptional activity. The receptor with bound steroid (termed the regulators, and the nitric oxide receptor, which is steroid receptor complex or liganded steroid receptor) soluble . These two systems are quite undergoes nuclear import, after which the liganded steroid different and represent the diverse mechanisms by which receptor dimerizes. The dimer binds its specific hormone animal cells respond to diffusible mediators that cross response element (HRE or SRE for steroid response the plasmalemma. While steroids alter gene and cell element) to activate or repress within 30 min about 50 regulation on a time scale of minutes to days, nitric to 100 primary response via its interactions with oxide (NO) has diverse effects (on a time scale of numerous other transcriptional factors and consequent seconds to hours) such as vasodilation, inhibition of changes in chromatin structure. Other secondary response platelet activation, , and cytotoxicity. In genes can then be regulated over the course of hours or days some instances, crosstalk exists between NO and steroid through mechanisms requiring expression of the primary receptor pathways. The and molecular response genes. The ability of a liganded steroid receptor to biology of steroids and NO can impact numerous find the handful of promoters containing HRE among the biotechnological applications such as gene therapy, tissue ~ 100,000 genes in the mammalian genome represents an engineering, and bioreactor dynamics, particularly in extraordinary diffusional search process. The dimer may relation to the external control of cellular undergo one-dimensional diffusion along DNA segments and gene expression. or DNA-intersegment transfer facilitated by the bivalent interactions made possible with a dimer complex (3). The steroid receptor has three functional domains: the STEROIDS AND THEIR BIOSYNTHESIS N-terminal domain (site of transcriptional activation and binding of transcriptional factors; A/B domain in Table 1), Steroid hormones are small hydrophobic metabolites a DNA binding domain (which also confers dimerization; of cholesterol (C27), and are broadly divided into five C/D domain in Table 1), and a hormone binding domain classes (Table 1): progestagens, , mineralo- at the C-terminus of the which also participates corticoids, androgens, and estrogens. Cleavage of the C6 in transcriptional activation (E domain in Table 1) (4). 1 17 Table 1. Steroids and their receptors Steroid Receptor Response element Steroid structure

Progestagens (C21) Dimerization Same as GRE Pregnenolone 17-hydroxypregnenolone Progesterone PR 17-hydroxyprogesterone DNA

transactivation Progesterone Glucocorticoids (C21) GR GRE Corticosterone 5'AGAACAnnnTGTTCT-3 Deoxycorticosterone Cortisol 11-Deoxycortisol Cortisone 18-hydroxy corticosterone 1-a-hydroxycorticosterone Aldosterone Cortisol

Mineralocorticoids (C21) MR Same as GRE Aldosterone

Aldosterone Androgens (C 19) AR Same as GRE 1 l-/*-hydroxyandrostene dione 5-a-Androstanediol Androsterone Epiandrosterone Andronosterone Testosterone Epitestosterone 5-a-dihydrotestosterone 5-/?-dihydrotestosterone 11-keto-testosterone

Estrogens (Cis) ER ERE 5'-PuGGTCAmInTGACCPy-^ Estrone Estriol

Estradiol

The DNA binding domain and hormone binding domain three-dimensional assemblies with general transcription are highly conserved across the steroid receptor family, factors (TBP, TAF//30, dTAF//110, TFIIB), coactivators while the N-terminus, which controls transcriptional (AEA70, RIP140, ERAP, TIF-2, SRC-I, RSP5, TIFl, regulatory activity, is not. Crystal structures are widely SUGl, GRIP170), sequence-specific transcriptional factors available for many mammalian steroid receptors (see (fos/jun, GATA-I, ReIA, PML, OTF-I), and chromatin http: 11nrr.georgetown.edu /NRR /NRR.html). regulators (hBrm, BRGl, Spt6, and HMGl) (5-11). The liganded steroid receptor dimer bound to its HRE Coactivators can bridge the dimer with other DNA binding has an extremely complicated and not fully characterized proteins of the transcriptional initation complex that set of interactions with other proteins. Once in the assemble on the TATA box of the promoter (Fig. 1). nucleus, receptors can interact in In contrast to activation, gene repression is commonly steroid hormone

hsp90,hsp70 hsp56, others

steroid CYTOPLASM receptor with caperones

NUCLEUS sequence-specific coactivators transcriptional activators transcription

chromatin HRE TATA structure

transcription initiation factors, RNA polymerase Figure 1. Steroid receptor function. viewed as an action of the steroid hormone dimer that: sequence are possible. A promoter may contain several (i) sterically blocks DNA binding sites used by other HREs. Receptors for glucocorticoids, , factors, such as fos/jun (API complex); (2) inactivates progesterone, and androgens utilize the same 6-base DNA normally competent transcription factors; or (3) serves as sequence 5'-AGAACA-S' (4). The utilizes a binding sink for factors needed for transcription (4). the sequence 5'-AGGTCA-S', which is also the sequence In addition to these issues of assembly on the promoter, used by nonsteroid nuclear receptors. The DNA binding steroid receptors may function by altering the structure domain of the steroid receptor contains two binding of the chromatin via its effects on hBrm and BRGl motifs —the P Box binds the HRE half-site and the D (5-7). hBrm is the homolog of Drosophila Box facilitates dimerization. Liganded steroid receptors Brama (brm) and SWI/SNF complex and is a predominantly form homodimers on the HRE in vivo. 180-kDa nuclear protein that mediates ATP-dependent The nonsteroidal thyroid hormone response elements nucleosome disruption. BRGl (205 kDa) is a second contain copies of a core consensus motif: 5'-AGGTCA- human homolog of yeast SWI/SNF that binds the 3' commonly in a head-to-tail repeat separated by four retinoblastoma gene product Rb, on which assembly base or inverted tail-to-tail repeats. Numerous variations processes occur. Interestingly, hBrm is not expressed in exist naturally or have been constructed artificially. The all cells. response elements for one or more hormone receptors may The human (PR) can bind steroid exist on a single promoter to cause synergistic activation receptor coactivator 1 (SRC-IA) for up-regulation of tran- of the gene. In promoter construct experiments, a scription. Interestingly, SRC-IA has histone acetyltrans- single glucocorticoid response element (GRE) (or estrogen ferase activity. Histone acetylation opens up the chromatin response element, ERE) upstream of a TATA box confers a structure for transactivating factors. Human PR can also steroid responsive promoter, with two GRE (or two ERE) bind p300/CREB-binding protein (CBP)-associated factor, elements resulting in synergistic gene activation. Such which has histone acetyltransferase activity. The localiza- experiments indicate that the steroid tion of histone acetyltransferase activity by PR suggests dimer can interact directly with TATA binding factors to an important role in chromatin reorganization since his- cause gene activation, independent of other transcriptional tone deacylase activity can repress the PR activation of factors. Mutations in steroid receptors and their associated gene expression (12). Similar interactions are found for proteins are implicated in a variety of developmental, the human estrogen receptor (13). inflammatory, and carcinogenic processes. For example, The HRE are typically palindromes containing two 6- the has a critical role in male sexual base consensus half-site sequences separated by a spacer differentiation, and its mutations may play a role in region (Table 1), although direct repeats of the consensus prostate (10). NITRIC OXIDE AND NITRIC OXIDE SYNTHASE Whiteley et al. — Protein Processing, Processing in the and Golgi Network). The pioneering discovery by R. Furchgott in 1980 (14) that -induced vasodilation was endothelial- dependent set the stage for the identification of NITRIC OXIDE: cGMP-DEPENDENT SIGNALING endothelial-derived relaxing factor (EDRF) as nitric oxide. By 1998, over 22,000 publications have defined in great Nitric oxide rapidly diffuses through cellular membranes detail the biochemistry, , and to bind its intracellular receptor, soluble guanylate cyclase, of the nitric oxide pathway. In mammalian cells, L- leading to the production of cGMP. Guanylate cyclase is a is cleaved by nitric oxide synthase (NOS) to NO and cit- -containing protein. Nitric oxide is a highly reactive rulline (Fig. 2). Three isoforms of NOS have been cloned species that exerts autocrine and paracrine action over and their gene products fully characterized: neuronal NOS very short distances of a few hundred micrometers due (nNOS, Type I isoform, 155 kDa); inducible NOS (iNOS, to its short half- in biological fluids. However, recent Type II isoform, 130 kDa); and endothelial NOS (eNOS, evidence has raised the possibility that NO can bind to Type III isoform, 140 kDa). The genes are highly homolo- oxyhemoglobin in an S-nitrosothiol complex (21). gous across species, and the three isoforms are about 50% Endothelial-produced NO, elicited by shear stress, can homologous with each other (15). For full NO synthesis act as an autocrine factor to elevate cGMP levels (22). activity, the eNOS requires three co-substrates, NO released by can diffuse through the L-arginine, O2, and NADPH, along with several cofac- internal elastic lamina to reach cells in the tors, FMN, FAD, (BH4), media of the vessel. In smooth muscle cells, the elevated and a heme group, as well as, superoxide dismutase and levels of cGMP caused by NO binding and activating glutathione. Distinct from iNOS, the isoforms eNOS and soluble guanylate cyclase results in a potent smooth nNOS are activated when intracellular calcium is elevated muscle cell relaxation and consequent vasodilation. The above resting levels. The eNOS gene does not contain a signaling pathway downstream of guanylate cyclase TATA box, which is not uncommon for constitutive genes. activation includes the activation of cGMP-dependent The eNOS promoter contains two AP-I sites, five SPl (GC- protein and subsequent phosphorylation of several rich) sites, and SSRE and NFl sites. The SP-I sites in the targets such as calcium-sensitive potassium channels eNOS promoter have been shown to be functional by gel (Kca)- Additionally, there exist cGMP-gated channels shift assays (16). The iNOS gene is dramatically induced and cGMP-regulated cyclic phosphodiesterases. in macrophages, smooth muscle cells, and other cell types by (IFN-y, TNF-a, and IL-I) as well as bacterial endotoxin. The transcriptional factor NF-KB is critical for NITRIC OXIDE: cGMP-INDEPENDENT SIGNALING the induction of iNOS. In several bioprocess or biomedical environments, the During the bioreactor cultivation of animal cells, sublytic cellular response to mechanical forces can regulate cellular fluid shear forces may have significant effects on cellu- function. Nitric oxide can play an important role in the lar metabolism. Hydrodynamic shear stress elevates the response of mammalian cells to fluid mechanical forces. release of NO and (PGI2) from endothelial The eNOS mRNA and protein levels can be elevated cells in culture (23,24). Recent reports have suggested several fold in endothelial cells when the cells are exposed that elevated levels of NO can lead to the activation to laminar shear stress (15 or 25 dyn/cm2) (17,18). The of constitutive (COXl, also named con- eNOS enzyme can be activated by fluid shear stress via stitutive PGH synthase 1) (25-27). Davidge et al. (25) a -dependent mechanism that is largely tested whether NO-activated PGI2 production required independent of elevation of intracellular calcium (19) cGMP using several different approaches. Their work indi- or binding of hsp90 (20). During chemical activation of cated that NO stimulation of PGI2 production is most endothelium, eNOS can be phosphorylated, palmitoylated, likely independent of cGMP levels. This is consistent and myristoylated at specific sites on the enzyme. In light with earlier reports that elevated levels of cGMP do not of eNOS activation by shear stress, several changes in enhance PGI2 production in bovine endothelial cells (28). the post-translational state of eNOS are expected, (see Treatment of bovine aortic endothelial cells in culture

Ca2+ Figure 2. The NO pathway. Activation of CaM ONOQ- tyrosine nitration nitric oxide synthase (NOS) results in the pro- O2 O " duction of nitric oxide (NO), which can bind and 2 cGMP-dep activate soluble guanylate cyclase (GC), result- cGMP ing in the production of cyclic GMP (cGMP). The NOS NO GC activation of cGMP-dependent protein kinase leads to phosphorylation events that alter cel- lular function. Through nonenzymatic routes, L-arginine citrulline NO rapidly reacts with superoxide anion (O2~) NADPH heme groups to form peroxynitrite (ONOO"), which can react thiols with tyrosine residues to form nitrotyrosine. NADP (BAEC) with glyceryl trinitrate, , or (ICAM-I) by shear stress (37) via elevated 3/-morpholinosydnonimine (SIN-I, a donor of NO and 02", production of ROS. Similarly, NAC impairs the induction and consequently peroxynitrite) dramatically enhanced of ICAM-I, monocyte chemotactic protein-1 (MCP-I), and by four- to six fold the release of PGI2 from arachidonic plasminogen activator inhibitor-1 (PAI-I) in endothelium acid-stimulated BAEC via cGMP-independent path- exposed to cyclic stretch (38-40). It is unclear why ROS ways (27). In calcium ionophore A23187-treated bovine generated by stretch (but not shear stress) can induce microvessel endothelial cells, both NO and PGI2 produc- the PAI-I gene. Interestingly, the MCP-I gene, which tion were increased several fold in a cGMP-independent is induced by shear stress via AP-I elements in the manner, but inhibition of NO production with NG-nitro- promoter (41) by mechanisms that likely involve ROS (38), L-arginine methyl ester (LNAME) attenuated the PGI2 is also induced by NO (42). The activation by shear stress release by half (25). Recently, peroxynitrite generated of AP-I activity, and shear stress mediated induction of from the reaction of NO with O2" was shown to acti- ICAM-I, MCP-I, and PAI-I through ROS pathways, may vate purified ram seminal vesicle COXl and recombinant involve NO and peroxynitrite. human C0X2 by serving as a substrate for the peroxi- Protein nitration by peroxynitrite has been hypothe- dase activity of the (29). While BAEC posseses a sized to be a major mechanism of oxidative modification soluble guanylate cyclase that can be activated by nitric of proteins associated with atherosclerosis (43). Peroxyni- oxide to elevate intracellular cGMP levels (22), there is trite the aromatic ring of free tyrosine to produce some uncertainty if human umbilical endothelial cells a stable product, 3'-nitrotyrosine, which accumulates in or BAEC possess cGMP-dependent protein kinases (30). proteins and acts as a cumulative index of peroxyni- However, a more recent report has demonstrated cGMP- trite production. This reaction occurs spontaneously but dependent protein kinase type I in BAEC (31). In light of is also catalyzed by low-molecular-mass transition metals the above observations, Wang and Diamond (32) tested as well as superoxide dismutase. This aberrant addition the hypothesis that shear induced nitric oxide was respon- of a nitrate group to the ortho position of the hydroxyl sible for the elevated release of PGI2 when endothelial group of tyrosine represents one outcome of cellular oxida- cells were exposed to arterial levels of shear stress. They tive stress, rendering nitrated proteins dysfunctional and found that blocking shear-induced NO production caused disrupting the phosphorylation of tyrosine residues in pro- ~50% decrease in shear-induced production of PGI2. teins involved in networks. More specifically, While the COX enzymes are potential targets for NO peroxynitrite was recently reported to have vasoregulatory activation because they contain an iron-heme center at significance by relaxing vascular tissue through nitrosy- their active site, Tsai et al. (33) found no activation of lation of tissue glutathione (or other thiols), subsequently cyclooxygenase activity at NO concentrations ranging releasing NO over prolonged time periods (44,45). from micromolar to millimolar added to purified ovine seminal vesicle COXl enzyme. This is in contrast to a CROSSTALK BETWEEN NITRIC OXIDE AND STEROID report of heme-independent S-nitrosation of cysteines in the active site of COXl (34). Furthermore, in the mouse SIGNALING macrophage cell line RAW264.7, lipophilic superoxide dismutase-mimetic agents decreased the The nuclear factor NF-/cB exists as an inactive cyto- production without affecting the level of NOS or COX or plasmic heterotrimer of p50 and p65 complex bound by inhibiting the release of (29). Clearly, by the inhibitor LcB. Upon phosphorylation of IKB by nitric oxide has diverse effects as an intracellular signaling tyrosine kinases or I/cB kinase [potentially activated by molecule, many of which are independent of soluble protein kinase C isoforms that are activated by shear guanylate cyclase. stress (46)], the heterodimer p50,p65 is released as an active DNA binding complex, which rapidly translocates to the nucleus. NF-/cB activity is inhibited by dexam- NITRIC OXIDE, PEROXYNITRITE, AND NITROTYROSINE ethasone, , and via distinct mechanisms (47). The expression of the iNOS gene is dra- Excessive production of nitric oxide has also been matically elevated by cytokines and endotoxin through associated with cellular injury (35). Nitric oxide-related a NF-/cB pathway. The induction of iNOS is markedly tissue injury may be at least partially due to peroxynitrite blocked by the steroid dexamethasone (48). Thus NF- (ONOO"), a relatively long-lived, strong cytotoxic oxidant KB and IKB regulation represents an important point of that is generated by the diffusion-limited reaction between crosstalk between NO/NOS and steroid pathways. Fur- nitric oxide (NO) and superoxide anion (02~) with a thermore, it has been shown that nitric oxide donors second-order rate constant of 6 x 109 M"1 sec"1 (36). High such as S--glutathione decrease induction levels of NO production can result in the formation of of endothelial vascular molecule (VCAM-I) significant amounts of ONOO", because nitric oxide is and endothelial-leucocyte adhesion molecule (ELAM) (49). capable of outcompeting superoxide dismutase for the Promoter construct experiments and gel shift assays metabolism of cellular superoxide. indicated that NO partially inhibited the activation of and (ROS) may NF-/cB. This is consistent with the role of NO as an anti- contribute to the response of mammalian cells to various inflammatory and antiatherosclerotic agent (50) similar environmental stimuli, either chemical or mechanical. The in function to steroids such as estrogen. While NF-/cB N-acetyl cysteine (NAC) has been shown to activity is induced at early times of shear stress expo- impair the induction of endothelial intracellular adhesion sure (51), probably through PKC and mitogen activated protein (MAP) kinase signaling, NF-/cB may eventually gene construct containing a modified HRE. Addition of be downregulated through the continued elevated produc- the steroid analog (that has no activity for or against the tion of NO by shear-stressed endothelium. There have endogenous receptor) would activate the modified receptor been several reports of endothelial genes (for example, that would then bind the novel HRE of the transgene PAI-I) being less inducible by cytokines after shear stress of interest. While chimeras utilizing the human steroid exposure (52). It is attractive to speculate that the lack binding domain and yeast transactivator GAL4 are useful of cytokine inducibility in sheared cells is NO dependent. tools of study, the expression of a yeast protein in This scenario, however, is in contrast to NO causing MAP would present likely immunological complications. Thus kinase and p21ras activation, and NF-/cB induction in the human steroid receptor gene requires mutation to , as well as activation of G proteins (53-55). confer novel ligand and DNA binding properties. Both Also, NO increases the amount of GTP-bound p21ras in rational design and combinatorial/screening strategies human T cells, likely through S- of a criti- may produce optimal steroid analog/receptor constructs cal cysteine in p21ras that enhances nucleotide that meet these goals. exchange (53-55). Finally, an important example of NO- The biology of nitric oxide may impact gene therapy, steroid crosstalk is the antiatherosclerotic effects of , and biocultivation dynamics of human estrogen. This activity of estrogen is ascribed to the cells. Nitric oxide synthase has been an important target enhanced production by endothelial cells of NO through for gene therapy of blood vessels to prevent restenosis estrogen-dependent inhibition of superoxide anion produc- after balloon angioplasty. Nitric oxide is widely viewed tion (56). as an inhibitor of smooth muscle (a major source of wall thickening) and an inhibitor of platelet SUMMARY: RELEVANCE TO BIOTECHNOLOGY and neutrophil activation and adhesion. The diffusible nature of nitric oxide in conjunction with its short half- life and local zone of action makes the eNOS gene an Numerous expression vectors utilize hormone response attractive candidate for gene therapy. Furthermore, a elements to confer external regulation of the gene major challenge in tissue engineering an artificial blood of interest. These vectors are used in bioprocessing vessel requires the creation of a nonthrombogenic lining. applications to time the induction of protein expression, The antiplatelet activity of nitric oxide is a primary independent of other growth-dependent phases of the motivation for seeding tissue constructs with endothelium bioreactor dynamics. In biotechnology applications where or engineered smooth muscle cells expressing nitric oxide expression of a gene is linked to the , it synthase. Thus nitric oxide, when locally produced at the is difficult to optimize simultaneously the cell growth correct level, may prevent intimal hyperplasia, accelerated rate, protein expression per cell, and overall reactor atherosclerosis, and thrombosis that would reduce the productivity. For example, expression of the heavy- patency of implanted grafts. Finally, in the biocultivation and light-chain mRNA by hybridomas may display cell- of complex human cells such as endothelium, stem cells, cycle dependency, with high expression during stationary or , the intracellular signaling generated through phase. The use of an HRE-rich promoter for these genes nitric oxide pathways may be an important consideration. would allow a precisely timed induction of the genes or This is especially true in cellular systems where the NO utilization of a continous process for protein expression, pathways are highly sensitive to chemical perturbations in thus avoiding batch configurations typical of hybridoma oxygen or glucose or to mechanical pertubations common technology. Furthermore, recent evidence of to bioreactors. activation of kinases that phosphorylate steroid receptors suggests important crosstalk between steroids and growth factors that may be exploited to improve bioreactor ACKNOWLEDGMENTS operation. In gene therapy, retroviral and adenoviral vectors can The work was supported by NIH Grant HL47486. The contain hormone response elements to help control the author is the recipient of the National Foundation tissue distribution, timing, and level of expression of the National Young Investigator Award no. BES9358236. therapeutic gene (57). Steroid-regulated promoters can be constructed with as little as 300 bp of DNA and thus can meet the packing constraints imposed by viral-based BIBLIOGRAPHY gene transfer. Additionally, the level of expression can be controlled by the level of the administered hormone 1. W.B. Pratt and D.O. Toft, Endocr. Rev. 18, 306-360 (1997). and/or the number of repeats of the response element 2. B.C. Freeman, D.O. Toft, and R.I. Morimoto, Science 274, in the promoter. A limitation of this approach is that 1718-1720(1996). wild-type genes would also be susceptible to the hormonal 3. B.A. Lieberman and S.R. Nordeen, J. Biol. 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