Human Uteroglobin Research Articles

Use of Uteroglobin for the Engineering of Polyvalent, Polyspecific Fusion Proteins

We report a novel strategy to engineer and express stable and soluble human recombinant polyvalent/polyspecific fusion proteins. The procedure is based on the use of a central skeleton of uteroglobin, a small and very soluble covalently linked homodimeric protein that is very resistant to proteolytic enzymes and to pH variations. Using a human recombinant antibody (scFv) specific for the angiogenesis marker domain B of fibronectin, interleukin 2, and an scFv able to neutralize tumor necrosis factor-alpha, we expressed various biologically active uteroglobin fusion proteins. The results demonstrate the possibility to generate monospecific divalent and tetravalent antibodies, immunocytokines, and dual specificity tetravalent antibodies. Furthermore, compared with similar fusion proteins in which uteroglobin was not used, the use of uteroglobin improved properties of solubility and stability. Indeed, in the reported cases it was possible to vacuum dry and reconstitute the proteins without any aggregation or loss in protein and biological activity.

Uteroglobin induces the development and cellular proliferation of the mouse early embryo

Two-cell mouse embryos cultured in vitro in the presence of either purified rabbit uteroglobin (UG) or recombinant human UG developed and proliferated faster than controls cultured in the absence of this protein. Both the percentage of embryos developing to the blastocyst stage and the number of cells per embryo were increased. Treatment with UG for 3 hr was enough to trigger this response. The effect of UG was blocked by genistein, an inhibitor of tyrosine protein kinases, suggesting the involvement of these kinases in the stimulation of the embryo by UG. To further support this suggestion, embryos were metabolically labeled in vitro with [32P] and the phosphorylated proteins were immunoprecipitated with anti-phosphotyrosine. Analysis of the immunoprecipitates by SDS-PAGE showed that UG induced the phosphorylation of several proteins of M(r) between 200 and 37 kDa. This induction was observed after 1 hr of stimulation with UG and further increased after 3 hr of treatment. Since UG is synthesized and secreted in the uterus and the oviduct, these results suggest a physiological role of this protein in the correct development of the embryo in vivo.

Recombinant human uteroglobin/CC10 inhibits the adhesion and migration of primary human endothelial cells via specific and saturable binding to fibronectin

Uteroglobin (UG) or Clara Cell 10 kDa protein (CC10) is a small, stable, epithelial secretory anti-inflammatory protein. Uteroglobin has been shown to inhibit neointimal formation in vivo after balloon angioplasty through an unknown mechanism. An interaction between UG and plasma fibronectin (Fn) has been demonstrated in mice. Since Fn plays a key role in endothelial cell (EC) migration and angiogenesis, we investigated whether recombinant human UG (rhUG) affects EC migration via Fn binding. In this report, we show a saturable binding of rhUG to Fn depending on Fn conformation and that rhUG is covalently cross-linked to Fn by transglutaminase (TGase). Additionally, our study highlights that rhUG can also bind to exogenously added or self-secreted Fn on the membrane of human primary microvascular endothelial cells (HMVEC), although these complexes are weakly associated with the plasmalemma. Upon the interaction with Fn in solid phase, rhUG strongly inhibits HMVEC attachment on Fn, but not on other ECM proteins. Consequently, rhUG also inhibits cell migration in a dose dependent fashion (I.C.50 = 65 nM) and hinders the "wound healing" in vitro. The small size, stability and human tolerability of rhUG suggest that rhUG in slow-release form or genetically delivered could be used in humans to modulate cell/Fn interactions in the context of tumor microenvironment or in the context of inflammation and fibrosis.

Association of the Human Uteroglobin Gene Polymorphism with Primary Lung Cancer

Association of the Human Uteroglobin Gene Polymorphism with Primary Lung Cancer

New weapons against inflammation: dual inhibitors of phospholipase A2 and transglutaminase.

Despite decades of research, corticosteroids and NSAIDs remain the main pharmacological weapons to control inflammation in the clinic. Unfortunately, these drugs have significant side effects, especially when used chronically. Consequently, there is tremendous interest in the development of novel, safer, and more effective anti-inflammatory drugs. Most anti-inflammatory agents directly or indirectly inhibit the formation or the effects of arachidonic acid metabolites collectively known as eicosanoids (prostaglandins, leukotrienes, thromboxanes, endoperoxides, and other mediators) (Table ​(Table1).1). It has long been recognized that inhibition of phospholipase A2–catalyzed (PLA2-catalyzed) arachidonic acid release from cell-membrane glycerophospholipids could potentially block the synthesis of all eicosanoids. The vast PLA2 enzyme family includes cellular isoforms involved in signal transduction, such as three cellular isoforms of PLA2 (cPLA2s), and ten secretory isoforms of PLA2 (sPLA2s) (1). Various sPLA2 isoforms participate in digestive physiology, antimicrobial defense, and inflammation. The cPLA2s, and sPLA2s IIA and V, play key roles in arachidonic-acid release during acute inflammation (1). Two families of endogenous proteins include members whose synthesis and/or secretion are induced by glucocorticoids in the lung that exhibit anti-inflammatory activity in experimental models. These are the lipocortins, or annexins (2), and the secretoglobins, whose prototype is uteroglobin (3). These families include proteins with distinct and pleiotropic biological properties. Lipocortins I and V, as well as rabbit and human uteroglobin, have anti-inflammatory properties that can be explained, at least in part, by their ability to inhibit sPLA2. Human uteroglobin or “Clara Cell 10 kDa protein” is currently in clinical development for the prevention of airway inflammation in neonatal lung disease. The mechanism of sPLA2 inhibition by lipocortins and uteroglobin remains controversial and may depend on the assay system. However, a 9–amino acid sequence that is highly conserved in uteroglobin and the anti-inflammatory lipocortins I and V was identified as early as 1988 (4). Synthetic peptides corresponding to this shared sequence exhibit striking anti-inflammatory activity in vivo and inhibit sPLA2 in vitro. Mutagenesis data show that this sequence is necessary for sPLA2 inhibition by uteroglobin (5). Peptides derived from uteroglobin and lipocortins are collectively known as antiflammins, now recognized as one of the most potent classes of anti-inflammatory agents identified to date (6). The elegant work by Sohn et al. (7) appearing in this issue of the JCI builds on that early discovery and on the observation that some sPLA2s are further activated by post-translational modifications catalyzed by transglutaminases (TGases). Transglutaminases are multifunctional enzymes that form isopeptide bonds between specific lysine and glutamine residues of substrate proteins or crosslink polyamines to glutamine residues (8). Cordella-Miele et al. showed that the TGase-catalyzed formation of an intramolecular isopeptide bond within sPLA2s (9) or the polyamination of sPLA2 (10) enhances the activity of sPLA2s. Basing their work on these findings, Sohn et al. designed a novel series of chimeric peptides that include a fragment of pro-elafin (a TGase substrate in keratinocytes), and the conserved core of antiflammins (the sequence KVLD corresponding to uteroglobin residues 43–46). These new peptides inhibit sPLA2 and TGase activity, and the TGase-catalyzed post-translational activation of sPLA2 (Figure ​(Figure1).1). Interestingly, the authors show that even the original antiflammins inhibit TGase, though not as efficiently as the new chimeric peptides. Uteroglobin is a well-known TGase substrate and Lys 43 a likely acyl acceptor (11). The chimeric peptides exhibit dramatic in vivo anti-inflammatory activity in a clinically relevant model of allergic inflammation: ragweed pollen–induced allergic conjunctivitis in guinea pigs. Inhibition of sPLA2 and TGase activity was documented in tissue extracts from treated animals, and in vivo anti-inflammatory activity correlated with in vitro inhibitory potency on sPLA2 and TGase. Chimeric peptide R2 was as potent as topical steroid or antihistamine drops, based on clinical inflammation scores, and was even more effective in reducing eosinophil infiltration. These findings have potentially great therapeutic relevance if one considers the number of patients who are chronically treated with antihistamines or steroids for seasonal allergies. Figure 1 sPLA2s hydrolyze the ester bond at the sn-2 position of membrane glycerophospholipids, generating free arachidonic acid. This acid is metabolized in a complex series of reactions involving COX or lipoxigenases (LOX), generating pro-inflammatory eicosanoids. ... Table 1 Mediators and inhibitors of eicosanoid synthesis and inflammation

Uteroglobin: A Potential Novel Tumor Suppressor and Molecular Therapeutic for Prostate Cancer

Currently, there are very few diagnostic or therapeutic strategies targeted at controlling tumor growth and progression towards metastasis. Uteroglobin (UG) is a naturally occurring, small, stable, secretory protein that is normally expressed by most cells of epithelial origin but is known to be lost during the progression of prostate, lung, and uterine cancers to invasive malignancy. Uteroglobin −/− knockout mice appear to be extremely cancer prone. Both pharmacological and transgenic reconstitution of recombinant human UG (rhUG) to prostate, lung, and endometrial tumor cell lines markedly inhibits their invasiveness and antagonizes the neoplastic phenotype. In preliminary studies, rhUG inhibited angiogenesis in the ex vivo rat aorta model and showed antitumor activity against human prostate tumor cells (PC-3) in the chick chorioallantoic membrane assay, reducing both tumor volume and vascularity. A recent in vivo pilot study showed that twice daily dosing with rhUG resulted in a statistically significant increase in survival without evidence of toxicity in severe combined immunodeficient mice challenged with a PC-3 cell metastasizing tumor. Thus, rhUG may slow the progression of cancer by inhibiting both tumor cell invasiveness and tumor angiogenesis. It therefore holds the potential to serve as a new weapon in the arsenal of cytostatic, antimetastatic, adjuvant treatment for cancer. In this paper, we will briefly discuss the therapeutic potential of uteroglobin-based strategies for managing prostate cancer.

Lys 43 and Asp 46 in α-helix 3 of uteroglobin are essential for its phospholipase A 2 inhibitory activity

Uteroglobin (UG) is an anti-inflammatory, secreted protein with soluble phospholipase A 2 (sPLA 2)-inhibitory activity. However, the mechanism by which UG inhibits sPLA 2 activity is unknown. UG is a homodimer in which each of the 70-amino acid subunits forms four α-helices. We previously reported that sPLA 2-inhibitory activity of UG may reside in a segment of α-helix 3 that is exposed to the solvent. In addition, it has been suggested that UG may inhibit sPLA 2 activity by binding and sequestering Ca ++, essential for sPLA 2 activation. By site-specific mutation, we demonstrate here that Lys 43 Glu, Asp 46 Lys or a combination of the two mutations in the full-length, recombinant human UG (rhUG) abrogates its sPLA 2-inhibitory activity. We demonstrate further that recombinant UG does not bind Ca ++ although when it is expressed with histidine-tag (H-tag) it is capable of binding Ca ++. Taken together our results show that: (i) Lys 43 and Asp 46 in rhUG are critical residues for the sPLA 2-inhibitory activity of UG and (ii) Ca ++-sequestration by rhUG is not likely to be one of the mechanisms responsible for its sPLA 2-inhibitory activity.

Crystal structure analysis of recombinant human uteroglobin and molecular modeling of ligand binding.

Uteroglobin, a steroid-inducible, cytokine-like, secreted protein with immunomodulatory properties, has been reported to bind progesterone, polychlorinated biphenyls (PCB), and retinol. Structural studies may delineate whether binding of ligands is a likely physiological function of human uteroglobin (hUG). We report a refined crystal structure of uncomplexed recombinant hUG (rhUG) at 2.5-A resolution and the results of our molecular modeling studies of ligand binding to the central hydrophobic cavity of rhUG. The crystal structure of rhUG is very similar to that of reported crystal structures of uteroglobins. Using molecular modeling techniques, the three ligands--PCB, progesterone, and retinol--were docked into the hydrophobic cavity of the dimer structure of rhUG. We undocked the progesterone ligand by pulling the ligand from the cavity into the solvent. From our modeling and undocking studies of progesterone, it is clear that these types of hydrophobic ligands could slip into the cavity between helix-3 and helix-3' of the dimer instead of between helix-1 and helix-4 of the monomer, as proposed earlier. Our results suggest that at least one of the physiological functions of UG is to bind to hydrophobic ligands, such as progesterone and retinol.

Amino acid residues in alpha-helix-3 of human uteroglobin are critical for its phospholipase A2 inhibitory activity.

Amino acid residues in alpha-helix-3 of human uteroglobin are critical for its phospholipase A2 inhibitory activity.

Human uteroglobin gene polymorphisms and genetic susceptibility to asthma.

Human uteroglobin gene polymorphisms and genetic susceptibility to asthma.

The discovery of uteroglobin and its significance for reproductive biology and endocrinology.

The discovery of uteroglobin resulted from investigations on the biochemical composition of oviductal and uterine secretions of the rabbit and other mammals. These determinations about physiological composition were urgently requested to prepare culture media for research on early mammalian development in vitro. Discovery of significant proteins during the sixties reflected the laboratory skills of that time. Protein characterization was achieved by isolation via Sephadex gels, electrophoresis on polyacrylamide gels, and finally immunoprecipitation using classical polyclonal antibodies. The molecular biology was not yet established. Uteroglobin could be found as the major protein component of rabbit uterine secretion. From the beginning, it was already identified as an unusually small, spheric uterine secretory molecule without any carbohydrates--hence its name. Uteroglobin was the first mammalian protein that turned out to be progesterone-regulated and, at the same time, released in mg amounts actually in one organ compartment. Moreover, uteroglobin and its gene proved to be a reliable model for the description of progesterone/progesterone receptor complex action at the DNA level. After its original observation in the uterus, however, uteroglobin was detected also in several other organs, for example, the epididymis, the seminal vesicle, and the lung. Initially, it could not be found in the blood, which challenged the hypothesis that uteroglobin specifically should operate by local activation rather than by a humoral or endocrine effect. Later, though, the human uteroglobin molecule, isolated from blood filtrate, was used for detailed structural analyses. The rabbit uteroglobin model certainly was beneficial for reproductive biological research. Experimental interference with steroid hormone regulation during preimplantation presented surprising effects, which led to the discovery of the transposition of the implantation window. The uterine secretion protein patterns, in particular the uteroglobin fraction and the beta-glycoprotein fraction, served as decisive marker profiles to identify the biological stage of the intrauterine microenvironment during preimplantation. This diagnostic procedure, using only protein parameters, enabled us to precisely predict the receptive stage of the endometrium for donated blastocysts to achieve implantation successfully.

Uteroglobin binding proteins: regulation of cellular motility and invasion in normal and cancer cells.

Uteroglobin (UG) is a multifunctional, secreted protein with anti-inflammatory and antichemotactic properties. While its anti-inflammatory effects, in part, stem from the inhibition of soluble phospholipase A2 (sPLA2) activity, the mechanism(s) of its antichemotactic effects is not clearly understood. Although specific binding of UG on microsomal and plasma membranes has been reported recently, how this binding affects cellular function is not clear. Here, we report that recombinant human UG (hUG) binds to both normal and cancer cells with high affinity (20-35 nM, respectively) and specificity. Affinity cross-linking studies revealed that 125I-hUG binds to the NIH 3T3 cell surface with two proteins of apparent molecular masses of 190 and 49 kDa, respectively. UG affinity chromatography yielded similar results. While both the 190- and 49-kDa proteins were expressed in the heart, liver, and spleen, the lung and trachea expressed only the 190-kDa protein. Some cancer cells (e.g., mastocytoma, sarcoma, and lymphoma) expressed both the 190- and 49-kDa proteins. Further, using functional assays, we found that UG dramatically suppressed the motility and extracellular matrix invasion of both NIH 3T3 and some cancer cells. In order to further characterize the anti-ECM-invasive properties of UG, we induced expression of hUG into cancer cell lines derived from organs that, under physiological circumstances, secrete UG at a high level. Interestingly, it has been reported that a high percentage of the adenocarcinomas arising from the same organs fail to express UG. Our results on induced hUG expression in these cells show that inhibition of motility and ECM invasion requires the expression of both UG and its binding proteins. Taken together, our data define receptor-mediated functions of UG in which this protein regulates vital cellular functions by both autocrine and paracrine pathways.

Uteroglobin reverts the transformed phenotype in the endometrial adenocarcinoma cell line HEC-1A by disrupting the metabolic pathways generating platelet-activating factor.

Uteroglobin, originally named blastokinin, is a protein synthesized and secreted by most epithelia, including the endometrium. Uteroglobin has strong anti-inflammatory properties that appear to be due, at least in part, to its inhibitory effect on the activity of the enzyme phospholipase A(2). In addition, recent experimental evidence indicates that uteroglobin exerts antiproliferative and antimetastatic effects in different cancer cells via a membrane receptor. The human endometrial adenocarcinoma cell line HEC-1A does not express uteroglobin. Thus, we transfected HEC-1A cells with human uteroglobin cDNA. The transfectants showed a markedly reduced proliferative potential as assessed by impaired plating efficiency as well as by reduced growth in soft agar. Cytofluorimetric analysis clearly indicated that in uteroglobin-transfected cells the time for completion of the cell cycle was increased. We previously demonstrated that HEC-1A cells actively synthesize platelet-activating factor, one of the products of phospholipase A(2) activity. In addition, we demonstrated that platelet-activating factor stimulates the proliferation of these cells through an autocrine loop. In uteroglobin transfectants, the activity of phospholipase A(2) and platelet-activating factor acetyl-transferase, which are involved in the synthesis of platelet-activating factor, was significantly reduced compared with wild-type and vector-transfected cells (p < 0.05). Our results indicate that enforced expression of uteroglobin in HEC-1A cells markedly reduced their growth potential and significantly impaired the synthesis of platelet-activating factor, an autocrine growth factor for these cells. These data suggest that one possible mechanism for the recently observed antineoplastic properties of uteroglobin may be the inhibition of the synthesis of platelet-activating factor.

Stable, long-term bacterial production of soluble, dimeric, disulfide-bonded protein pharmaceuticals without antibiotic selection.

Numerous biopharmaceuticals and other recombinant biotechnology products are made in prokaryotic hosts. However, bacterial production of native, biologically active eukaryotic proteins is rarely possible for disulfide-bonded and/or multisubunit proteins. We previously described the production of soluble, native disulfide-bonded dimeric proteins in the Escherichia coli cytoplasm (Miele et al., 1990; Mantile et al., 1993). Native, biologically active proteins with up to six disulfide bonds have been produced with our expression system (Garces et al., 1997). However, plasmid instability during induction limited its usefulness. We now report the stable, high-level expression of soluble, disulfide-bonded human uteroglobin without antibiotic selection. We designed a new vector containing a multifunctional stabilization region that confers complete plasmid stability and increased protein yields without copy number increases. Recombinant expression remains fully inducible after long-term continuous culture in nonselective liquid medium (at least 260 generations). This system may significantly expand the applications of bacterial expression to recombinant production of soluble, bioactive proteins for biochemical studies and biopharmaceutical/industrial purposes. As a result of the very broad activity spectrum of the stabilization region we selected, its use could be extended to bacterial hosts other than enterobacteria.

Expression of uteroglobin in the human endometrium.

Uteroglobin is a progesterone binding protein, a member of the antiflammin gene family and possibly a novel cytokine. Initially, uteroglobin was identified as the major protein of rabbit uterine secretion during the phase of preimplantation. Counterparts of the rabbit uteroglobin or its gene are described in rat, mouse, hamster, hare, pig, horse and human. While uteroglobin appears as one of the most extensively studied proteins, particularly its physico-chemical properties, including its crystal structure and its gene, the true physiological role of this protein still remains to be unravelled. Essential to understanding the significance of human uteroglobin in reproductive organs, particularly in the endometrium, is a knowledge of the spatial and chronological expression of this secretory protein. Our studies on 115 volunteers combined reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry and quantitative assessment by an enzyme-linked immunosorbent assay for uteroglobin. The expression, localization and release of uteroglobulin in the human endometrium are presented. Secretory uteroglobin is found in endometrial tissue homogenates in highest levels of expression during the early luteal phase (days 15-19, 340 pg/mg total protein). In turn, uteroglobin is released into the uterine lumen in peak amounts during the receptive phase of the menstrual cycle (mid-luteal phase, days 20-23, secretion level 833.4 pg/mg total protein). Our immunohistochemical studies match with these results, as uteroglobin is located during the early and mid-luteal phase in the apical compartments of endometrial gland cells. These observations strongly suggest an involvement of uteroglobin in endometrial preparations for implantation.

Uteroglobin: a novel cytokine?

Blastokinin or uteroglobin (UG) is a steroid-inducible, evolutionarily conserved, multifunctional protein secreted by the mucosal epithelial of virtually all mammals. It is present in the blood and in other body fluids including urine. An antigen immunoreactive to UG antibody is also detectable in the mucosal epithelia of all vertebrates. UG-binding proteins (putative receptor), expressed on several normal and cancer cell types, have been characterized. The human UG gene is mapped to chromosome 11q12.2 13.1, a region that is frequently rearranged or deleted in many cancers. The generation of UG knockout mice revealed that disruption of this gene causes: (i) severe renal disease due to an abnormal deposition of fibronectin and collagen in the glomeruli; (ii) predisposition to a high incidence of malignancies; and (iii) a lack of polychlorinated biphenyl binding and increased oxygen toxicity in the lungs. The mechanism(s) of UG action is likely to be even more complex as it also functions via a putative receptor-mediated pathway that has not yet been clearly defined. Molecular characterization of the UG receptor and signal transduction via this receptor pathway may show that this protein belongs to a novel cytokine/chemokine family.

Loss of transformed phenotype in cancer cells by overexpression of the uteroglobin gene.

Uteroglobin (UG) is a multifunctional, secreted protein that has receptor-mediated functions. The human UG (hUG) gene is mapped to chromosome 11q12.2-13.1, a region frequently rearranged or deleted in many cancers. Although high levels of hUG expression are characteristic of the mucosal epithelia of many organs, hUG expression is either drastically reduced or totally absent in adenocarcinomas and in viral-transformed epithelial cells derived from the same organs. In agreement with these findings, in an ongoing study to evaluate the effects of aging on UG-knockout mice, 16/16 animals developed malignant tumors, whereas the wild-type littermates (n = 25) remained apparently healthy even after 11/2 years. In the present investigation, we sought to determine the effects of induced-expression of hUG in human cancer cells by transfecting several cell lines derived from adenocarcinomas of various organs with an hUG-cDNA construct. We demonstrate that induced hUG expression reverses at least two of the most important characteristics of the transformed phenotype (i.e., anchorage-independent growth on soft agar and extracellular matrix invasion) of only those cancer cells that also express the hUG receptor. Similarly, treatment of the nontransfected, receptor-positive adenocarcinoma cells with purified recombinant hUG yielded identical results. Taken together, these data define receptor-mediated, autocrine and paracrine pathways through which hUG reverses the transformed phenotype of cancer cells and consequently, may have tumor suppressor-like effects.

Solution structure of the recombinant oxidized rabbit uteroglobin using homonuclear and heteronuclear multidimensional NMR.

Rabbit uteroglobin (rab-UG) is a 16-kDa homodimeric secretory protein with potent anti-inflammatory/immunomodulatory properties. Its physiological role is still unclear, although it was observed that several small hydrophobic molecules bind to the oxidized and the reduced uteroglobin. It is suggested that the formation and/or disruption of the two disulphide bridges not only regulates this binding itself, but also the affinity to the ligand. The determination of the solution structure has been started with the assignment of 1H, 15N and 13C resonances of the oxidized rabbit uteroglobin, based on several two-dimensional and three-dimensional homonuclear and heteronuclear double and triple resonance experiments. The assignment was possible with the overproduction of the wild-type as well as of uniformly 15N-labeled and 15N/13C-labeled samples of the recombinant protein. A complete assignment of 1H, 15N and 13C resonances, the secondary-structure elements and the tertiary structure in solution is presented. The tertiary solution structure was found to be in good agreement with the previously determined crystal structure of rab-UG and with the solution structure of human uteroglobin (h-UG). h-UG and rab-UG are extremely stable proteins within a wide range of pH and temperatures. Some of the binding characteristics of ligands of rab-UG and a mutant with all cysteine residues exchanged to serine residues are discussed.

Uterine flushing levels of CC-16, the putative human uteroglobin in a subfertile population: preliminary findings

Uterine flushing levels of CC-16, the putative human uteroglobin in a subfertile population: preliminary findings

Lipophilin, a novel heterodimeric protein of human tears

We identified a novel heterodimeric protein, lipophilin AC, in human tears. One of its components, lipophilin A (69 residues; mass, 7575.1; p I, 9.47) was homologous to the C1 and C2 components of prostatein (`estramustine-binding protein'), the major secreted protein of rat prostate. Human lipophilin C (77 residues; mass, 8854.1; p I, 4.94) was homologous to the rat prostatein C3 component and to human mammaglobin, a protein overexpressed in some mammary carcinomas. Tear lipophilins A and C expand the roster of human uteroglobin superfamily members and provide models for exploring these typically steroid-regulated and steroid-binding molecules.