Differential effect of small ubiquitin-like modifier (SUMO)-ylation of the androgen receptor in the control of cooperativity on selective versus canonical response elements.

The androgen receptor (AR) can be small ubiquitin-like modifier (SUMO)-ylated in its amino-terminal domain at lysines 385 and 511. This SUMO-ylation is responsive to several agonists, but is not induced by the pure antagonist hydroxyflutamide. We show that the main site of interaction of Ubc9, the SUMO-1 conjugating enzyme, resides in transcription activation unit 5. Overexpression of SUMO-1 represses the AR-mediated transcription, and this effect is abolished after mutating both SUMO-1 acceptor sites. On the other hand, the mutation of lysine 385 clearly affects the cooperativity of the receptor on multiple hormone response elements. Lysine 511 is not implicated in this function. Surprisingly, these effects on cooperativity clearly depend on the nature of the response elements. When selective androgen response elements, which are organized as direct repeats of 5'-TGTTCT-3'-like sequences, were tested, the lysine 385 mutation did not increase the androgen response. Point mutations changing the direct-repeat elements into inverted-repeat elements restored the effects of the lysine 385 mutation on cooperativity. In conclusion, SUMO-ylation of the AR might have a differential function in the control of cooperativity, depending on the conformation of the AR dimer bound to DNA.

Androgen specificity of a response unit upstream of the human secretory component gene is mediated by differential receptor binding to an essential androgen response element.

The expression of secretory component (SC), the epithelial receptor for poly-immunoglobulins, is regulated in a highly tissue-specific manner. In several tissues, e.g. lacrimal gland and prostate, SC synthesis is enhanced by androgens at the transcriptional level. In this study, we describe the presence of an androgen response unit, located 3.3 kb upstream of the sc transcription initiation site and containing several 5'-TGTTCT-3'-like motifs. Although each of these elements is implicated in the enhancer function, one element, the ARE1.2 motif, is found to be the main interaction site for the androgen receptor as demonstrated in in vitro binding assays as well as in transient transfection assays. A high-affinity binding site for nuclear factor I, adjacent to this ARE, is also involved in the correct functioning of the sc upstream enhancer. The ARE1.2 motif consists of an imperfect direct repeat of two core binding elements with a three-nucleotide spacer and therefore constitutes a nonconventional ARE. We demonstrate that this element displays selectivity for the androgen receptor as opposed to glucocorticoid receptor both in in vitro binding assays and in transfection experiments. Mutational analysis suggests that the direct nature of the half-site repeat is responsible for this selectivity. We have thus determined a complex and androgen-specific response unit in the far upstream region of the human SC gene, which we believe to be involved in its androgen responsiveness in epithelial cells of different organs such as prostate and lacrimal gland. We were also able to demonstrate that the primary sequence of a single nonconventional ARE motif within the enhancer is responsible for its androgen specificity.

The first exon of the human sc gene contains an androgen responsive unit and an interferon regulatory factor element.

Secretory component (SC) plays a key role in the transport of IgA and IgM to the lumina of many glands. The gene is constitutively expressed, but can be modulated by hormonal and immunological stimuli. Recently, the promoter and the first exon of the human sc gene have been cloned. The first exon contains a putative androgen/glucocorticoid response element (ARE/GRE) and an Interferon Regulatory Factor Element (IRF-E). Here we show that the ARE/GRE can bind the DNA-binding domain (DBD) of both the androgen (AR) and glucocorticoid receptor (GR) with a preference for the AR-DBD. In transient transfection experiments, this element confers higher responsiveness to androgens than to glucocorticoids. The IRF-E can function as an IRF-2, but surprisingly not as an IRF-I responsive element. We postulate that these two regulatory elements play a key role in the complex regulation of the sc gene in vivo.

Leukocyte migration and activation by murine chemokines.

Chemokines are a family of chemotactic cytokines which attract different types of leukocytes. This property, combined with some additional inflammatory and growth-regulatory activities, demonstrate their crucial role in the immune system. Chemokines are low molecular weight proteins and possess a typical positioning of four conserved cysteines. This family is further subdivided in two subfamilies depending on whether the first two cysteines are adjacent or not (CC and CXC chemokines, respectively). The CXC chemokines (including interleukin-8) predominantly attract neutrophils, whereas CC chemokines induce migration of monocytes, as well as other leukocyte cell types. In this article, the general characteristics of chemokines are reviewed. Furthermore, the murine CC chemokines, JE/MCP-1, MCP-3/MARC, MIP-1 alpha, MIP-1 beta, RANTES, TCA3, C10/MRP-1, MRP-2, and eotaxin, are discussed more in detail.

Selective DNA binding by the androgen receptor as a mechanism for hormone-specific gene regulation.

Steroid hormones control many physiological processes by activating specific receptors that act as transcription factors. In vivo, each of these receptors has a specific set of target genes, but in vitro the glucocorticoid, progesterone, mineralocorticoid and androgen receptors (class I receptors) all recognise response elements which are organised as inverted repeats of 5'-TGTTCT-3'-like sequences with a three nucleotide spacer. This poses the question how the in vivo specificity of the different steroid responses is mediated. To unravel the mechanisms involved, we have compared the structural features of the androgen-selective enhancers of the probasin, the secretory component and the sex-limited protein genes with those of non-selective enhancers in the mouse mammary tumour viral promoter and the C3(1) gene. The probasin promoter contains an androgen response element which is recognised with high affinity by the androgen receptor, but not by the other class I receptors. Swapping experiments between the DNA-binding domains of the androgen and glucocorticoid receptor revealed that it is not the first zinc finger, but rather the second zinc finger and part of the hinge region which contribute to this specificity. Three AR-specific aminoacids are involved in the probasin ARE recognition, but not in the C3(1) ARE binding by the AR. The location of these residues strongly suggests that an alternative dimerisation interface is involved in the probasin ARE binding. We could subsequently demonstrate that the AR binds direct repeats of 5'-TGTTCT-3'-like sequences in gel retardation assays as well as in transfection experiments. Moreover, the androgen-specific enhancers all contain direct repeats, and point mutations that change the nature of these elements into inverted repeats result in a change of specificity. It seems, therefore, that direct repeat elements can be the determinants of the AR-specificity. It will be exciting to learn how such DNA elements will affect the properties of the receptor dimer with respect to ligand binding, interactions between the aminoterminal domain and the ligand-binding domain, the recruitement of co-activators and cooperativity with other transcription factors.

Tissue-specific androgen responses in primary cultures of lacrimal epithelial cells studied by adenoviral gene transfer.

The lacrimal gland secretes most of the water and many proteins present in tear fluid. The composition of the tear fluid is affected dramatically by androgens, an observation which has been linked to the fact that more than 90% of the patients with Sjögren syndrome are female. Although the presence of androgen receptors in the lacrimal gland has been established, the molecular biology of the protective effects of androgens remains largely unknown. Here, we report the use of primary cultures of the lacrimal gland which express endogenous proteins under androgen control, as a more homologous test system for tissue-specific transcription studies. Infection with recombinant adenoviral vectors was the most efficient method to introduce foreign gene constructs in these cultures. A thus introduced mouse mammary tumor virus promoter was inducible with androgens and this effect was independent of the sexual genotype of the infected cells. By use of two recombinant adenoviral vectors containing genomic fragments of the SC gene, which is androgen responsive in the lacrimal gland, we could demonstrate the functionality of the sc promoter as well as its androgen regulation in this culture system.

Isolation of a lymphocyte chemotactic factor produced by the murine thymic epithelial cell line MTEC1: identification as a 30 kDa glycosylated form of MCP-1.

A medullary-type murine thymic epithelial cell line (MTEC1) established in our laboratory constitutively produces multiple species of chemotactic factors, which attract lymphocytes, neutrophils, and monocytes. Chemotactic proteins were isolated from MTEC1 supernatant and purified to homogeneity by adsorption to controlled pore glass, heparin-Sepharose chromatography, cation-exchange FPLC and RP-HPLC. A chemotactic factor for both lymphocytes and monocytes was identified as a 30 kDa protein by SDS-PAGE analysis under reducing conditions. After cleavage of the NH2-terminally blocked protein with formic acid, the amino acid sequence of the internal fragment was analysed and found to be identical to the amino acid sequence of mouse MCP-1/JE. The protein is hence identified as a glycosylated natural form of MCP-1/JE secreted by thymic epithelial cells. The 30 kDa glycosylated form of MCP-1 shows lower specific chemotactic activity (CA) for both lymphocytes and monocytes than the 6-7 kDa unglycosylated form of MCP-1.

Androgen receptor knockout and knock-in mouse models.

Androgens play an important role in male reproductive development and function. These steroid hormones mediate their actions by binding to the androgen receptor (AR). Diseases such as androgen insensitivity syndrome, prostate cancer, Kennedy's disease, and infertility can be caused by mutations in the AR. To get a better insight into the molecular working mechanisms of the AR, several knockout and knock-in mouse models have been developed. These models are reviewed here and are compared with human diseases.

The androgen receptor DNA-binding domain determines androgen selectivity of transcriptional response.

The AR (androgen receptor) is a hormone-dependent transcription factor that translates circulating androgen hormone levels into a physiological cellular response by directly regulating the expression of its target genes. It is the key molecule in e.g. the development and maintenance of the male sexual characteristics, spermatocyte production and prostate gland development and growth. It is also a major factor in the onset and maintenance of prostate cancer and a first target for pharmaceutical action against the further proliferation of prostate cancer cells. The AR is a member of the steroid hormone receptors, a group of steroid-inducible transcription factors sharing an identical consensus DNA-binding motif. The problem of how specificity in gene activation is achieved among the different members of this nuclear receptor subfamily is still unclear. In this report, we describe our investigations on how the AR can specifically activate its target genes, while the other steroid hormone receptors do not, despite having the same consensus monomeric DNA-binding motif. In this respect, we describe how the AR interacts with a newly identified class of steroid-response elements to which only the AR and not, for example, the glucocorticoid receptor can bind.

Change of specificity mutations in androgen-selective enhancers. Evidence for a role of differential DNA binding by the androgen receptor.

The androgen and glucocorticoid receptors recognize identical DNA motifs, leaving unanswered the question of how steroid specificity of transcriptional regulation is established in cells containing both receptors. Here, we provide evidence that subtle differences in low affinity DNA recognition might be a crucial element in the generation of steroid-specific responses. Here we identify simple hormone response elements in the mouse sex-limited protein enhancer and the human secretory component androgen response unit to be essential for the androgen specificity of both enhancers. We describe specific in vitro binding to these motifs by the DNA-binding domain of the androgen but not the glucocorticoid receptor. Both elements can be considered partial direct repeats of the 5'-TGTTCT-3' core binding motif. In addition, we show that specific point mutations in their left half-sites, essentially changing the nature of the repeats, strongly enhance the glucocorticoid sensitivity of the respective enhancers, whereas they have no effect on their androgen responsiveness. Accordingly, these mutations allow specific binding of the glucocorticoid receptor DNA-binding domain to both elements in vitro. With these experiments, we demonstrate that differential recognition by the androgen receptor of nonconventional steroid response elements is, at least in some cases, an important mechanism in androgen-specific transcriptional regulation.

Androgen-receptor-specific DNA binding to an element in the first exon of the human secretory component gene.

Androgens and glucocorticoids are steroid hormones, which exert their effects in vivo by binding and activating their cognate receptors. These intracellular receptors are transcription factors that can bind specific DNA sequences, called hormone response elements, located near the target genes. Although the androgen receptor (AR) and the glucocorticoid receptor (GR) bind the same consensus DNA sequence, androgen-specific responses can be achieved by non-conventional androgen response elements (AREs). Here we determine the specificity mechanism of such a selective element recently identified in the first exon of the human gene for secretory component (sc ARE). This sc ARE consists of two receptor-binding hexamers separated by three nucleotides. The DNA-binding domains of the AR and GR both bind the sc ARE, but, although the AR fragment dimerizes on the element, the GR fragment does not. Comparing the affinities of the DNA-binding domains for mutant forms of the sc ARE revealed that dimeric GR binding is actively excluded by the left hexamer and more precisely by the presence of a G residue at position -3, relative to the central spacer nucleotide. Inserting a G at this position changed a non-selective element into an androgen-selective one. We postulate that the AR recognizes the sc ARE as a direct repeat of two 5'-TGTTCT-3'-like core sequences instead of the classical inverted repeat. Direct repeat binding is not possible for the GR, thus explaining the selectivity of the sc ARE. This alternative dimerization by the AR on the sc ARE is also indicated by the DNA-binding characteristics of receptor fragments in which the dimerization interfaces were swapped. In addition, the flanking and spacer sequences seem to affect the functionality of the sc ARE.

Identification of mouse granulocyte chemotactic protein-2 from fibroblasts and epithelial cells. Functional comparison with natural KC and macrophage inflammatory protein-2.

Neutrophil and monocyte chemotactic factors were isolated from conditioned media of mouse fibroblasts and epithelial cells. Neutrophil chemotactic activities were purified to homogeneity using a four-step chromatographic procedure, and the corresponding proteins were identified by amino acid sequence analysis. Natural forms of the murine chemokines KC and macrophage inflammatory protein-2 were isolated from virus-infected fibroblasts. However, the major neutrophil chemotactic activity from fibroblasts stimulated with endotoxin plus double-stranded RNA and from PMA-treated epithelial cells resided in other 7- and 8-kDa proteins. Amino acid sequence analysis revealed a novel Cys-Xaa-Cys chemokine structure, characterized by the conservation of four cysteines and the Glu-Leu-Arg motif. Based on the completely identified primary structure of this natural protein, this chemokine must be considered to be the murine homologue of human and bovine granulocyte chemotactic protein-2 (GCP-2; 61 and 64% identical residues, respectively). Due to NH2-terminal cleavage, 11 different forms of mouse GCP-2 were discovered. In contrast to human and bovine GCP-2, functional comparison of long and short NH2-terminal forms of mouse GCP-2 demonstrated that truncated mouse GCP-2 (short form) has a higher specific activity in neutrophil activation (gelatinase B release) and chemotaxis assays. Furthermore, mouse GCP-2 was more potent than human GCP-2 on human neutrophils, and more active than murine KC and macrophage inflammatory protein-2 on mouse neutrophils. In view of the absence of a murine homologue for IL-8, NH2-terminally processed GCP-2 can be considered a major neutrophil chemoattractant in the mouse during the inflammatory response.

Characterization of synthetic human granulocyte chemotactic protein 2: usage of chemokine receptors CXCR1 and CXCR2 and in vivo inflammatory properties.

Human granulocyte chemotactic protein 2 (GCP-2) has originally been isolated from cytokine-stimulated osteosarcoma cells as a chemokine coproduced in minute amounts together with interleukin 8. Human GCP-2 (75 residues) was synthesized on a 0.25-mmol scale using Fmoc chemistry. After disulfide bridge formation and purification, monomeric GCP-2 was recovered as a 6-kDa protein; the pure synthetic protein showed a molecular mass of 8076 Da as determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The exact amino acid sequence of synthetic GCP-2 was confirmed by Edman degradation. Synthetic GCP-2 was an equally active (minimal effective concentration of 1-3 nM) chemoattractant for neutrophilic granulocytes as was natural 75-residue GCP-2. At concentrations up to 30 nM, synthetic GCP-2 did not stimulate eosinophil, monocyte, or lymphocyte chemotaxis. GCP-2 induced a dose-dependent increase in [Ca2+]i in neutrophils, 1 nM being the minimal effective concentration. The GCP-2-induced [Ca2+]i increase was completely prevented by pertussis toxin. Prestimulation of neutrophils with equimolar concentrations of purified natural IL-8, GROalpha, GROgamma and ENA-78 abolished the [Ca2+]i increase in response to 1 nM GCP-2. Alternatively, the [Ca2+]i rise induced by these CXC chemokines was inhibited by pretreatment of neutrophils with GCP-2. GCP-2 stimulated [Ca2+]i increases in CXCR1- and CXCR2-transfected cells, demonstrating that GCP-2 binds to both IL-8 receptors. Intradermal injection of synthetic GCP-2 resulted in a dose-dependent neutrophil accumulation and plasma extravasation in rabbit skin. To provoke this skin reaction, GCP-2 (10 pmol/site) was nearly as effective as IL-8, indicating that it is an important complementary mediator of the inflammatory response.

Cloning, bacterial expression and biological characterization of recombinant human granulocyte chemotactic protein-2 and differential expression of granulocyte chemotactic protein-2 and epithelial cell-derived neutrophil activating peptide-78 mRNAs.

Human osteosarcoma cells secrete a novel C-X-C chemokine called granulocyte chemotactic protein-2 (GCP-2), which was previously identified by amino acid sequencing of the purified natural protein. In order to understand the role of this new protein in inflammatory reactions, we cloned GCP-2 DNA sequences to generate recombinant protein and specific DNA probes and primers. By means of PCR on cloned cDNA of osteosarcoma cells induced by interleukin-1 beta and fibroblasts induced by lipopolysaccharide plus dsRNA, the complete coding domain of GCP-2 was isolated. This sequence was cloned into the bacterial expression vector pHEN1 and, after induction, GCP-2 was secreted into the periplasm of Escherichia coli. Recombinant GCP-2 (rGCP-2) was purified and characterized by SDS/PAGE as a monomeric 6.5-kDa protein and by amino-terminal sequencing. The chemoattractive potency of GCP-2 for neutrophilic granulocytes was about 10-times less than that of interleukin-8 and the minimal effective dose was 10 ng/ml. However, at optimal dose (100 ng/ml) the maximal chemotactic response was comparable with that of interleukin-8. Both characteristics correspond with those of natural GCP-2. In addition, intracellular calcium release in neutrophils by recombinant GCP-2 was achieved with as little as 10 ng/ml. Quantitation studies using reverse transcriptase and the polymerase chain reaction revealed higher GCP-2 mRNA production in normal fibroblasts than in tumor cells. When compared with epithelial-cell-derived neutrophil-activating peptide-78 (ENA-78) mRNA, the GCP-2 mRNA levels were higher in all cell lines tested. In addition, GCP-2 and ENA-78 expression seem to be differentially regulated in that phorbol ester and lipopolysaccharide have opposing effects on their mRNA induction in diploid fibroblasts and epithelial cells, respectively. Interleukin-1 was demonstrated to be a general inducer for both chemokines, while interferon-gamma down-regulates their mRNA expression. The availability of recombinant GCP-2 together with the quantitation studies on mRNA expression will help to further elucidate the biological role of GCP-2 during the inflammatory response.