Steroid sulfation and desulfation are fundamental pathways vital for a functional vertebrate endocrine system. After biosynthesis, hydrophobic steroids are sulfated to expedite circulatory transit. Target cells express transmembrane organic anion-transporting polypeptides that facilitate cellular uptake of sulfated steroids. Once intracellular, sulfatases hydrolyze these steroid sulfate esters to their unconjugated, and usually active, forms. Because most steroids can be sulfated, including cholesterol, pregnenolone, dehydroepiandrosterone, and estrone, understanding the function, tissue distribution, and regulation of sulfation and desulfation processes provides significant insights into normal endocrine function. Not surprisingly, dysregulation of these pathways is associated with numerous pathologies, including steroid-dependent cancers, polycystic ovary syndrome, and X-linked ichthyosis. Here we provide a comprehensive examination of our current knowledge of endocrine-related sulfation and desulfation pathways. We describe the interplay between sulfatases and sulfotransferases, showing how their expression and regulation influences steroid action. Furthermore, we address the role that organic anion-transporting polypeptides play in regulating intracellular steroid concentrations and how their expression patterns influence many pathologies, especially cancer. Finally, the recent advances in pharmacologically targeting steroidogenic pathways will be examined.

Nitric oxide (NO) production by the vascular endothelium maintains an essential antiinflammatory, cytoprotective influence on the blood vessel wall. A key component of this activity is attributed to prevention of leukocyte-endothelial cell interactions, yet the underlying mechanisms remain unclear. The NO receptor, soluble guanylate cyclase (sGC), is expressed in endothelial cells but fulfils an unknown function. Therefore, we used intravital microscopy in mesenteric postcapillary venules from WT and endothelial nitric oxide synthase (eNOS) knockout (eNOS ؊/؊ ) mice, and an sGC activator (BAY 41-2272), to investigate a potential role for sGC in the regulation of adhesion molecule expression and leukocyte recruitment. Leukocyte rolling and adhesion was 6-fold greater in eNOS ؊/؊ than WT animals. BAY 41-2272 and the NO-donor, diethylamine-NONOate, reduced leukocyte rolling and adhesion in eNOS ؊/؊ mice to levels observed in WT animals. These effects were blocked by the sGC inhibitor ODQ [1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one], which itself caused a 6-fold increase in leukocyte rolling and adhesion in WT mice. Increased leukocyte rolling and adhesion in IL-1␤-treated mice was also inhibited by BAY 41-2272. Fluorescence-activated cell sorting analysis in vitro and a specific P-selectin neutralizing antibody in vivo revealed that selective down-regulation of P-selectin expression accounted for the antiadhesive effects of sGC activation. These data demonstrate that sGC plays a key antiinflammatory role by inhibiting P-selectin expression and leukocyte recruitment.endothelium ͉ intravital microscopy T he recruitment of immune cells to sites of tissue injury is an important facet of an inflammatory response and is thought to represent a multistage process involving leukocyte rolling, adhesion, and emigration. Each stage of this response is triggered by the expression of specific adhesion molecules on the surface of leukocytes, platelets, and the vascular endothelium (see ref. 1). The regulation of this process therefore plays a key role in the development of an inflammatory response; moreover, it is clear that the active suppression of leukocyte͞platelet activation under physiological conditions is paramount for maintenance of blood vessel integrity and patency. Nitric oxide (NO) production by the vascular endothelium exerts an important cytoprotective, antithrombotic influence on the blood vessel wall by preventing the activation and adherence of circulating cells and platelets (2-4). Loss of this inhibitory effect of NO, and other endothelium-derived mediators including prostacyclin (PGI 2 ), results in a change in the endothelial cell to a prothrombotic, proinflammatory phenotype. It is thought that such changes contribute to diseases such as sepsis and atherosclerosis, and restenosis after balloon angioplasty. Hence, therapeutic intervention to prevent leukocyte͞platelet activation may be beneficial in numerous inf lammatory cardiovascular pathologies.The mechanism underlying the antiplatelet activity of NO ha...

We have identified a Factor VIII (FVIII) binding domain residing within the amino-terminal 272 amino acid residues of the mature von Willebrand Factor (vWF) subunit. Two dimensional crossed immunoelectrophoresis showed direct binding of purified human FVIII to purified human vWF. After proteolytic digestion of vWF with Staphylococcus aureus V8 protease, FVIII binding was seen only with the amino-terminal SP fragment III and not with the carboxy-terminal SP fragment II. A monoclonal anti-vWF antibody (C3) partially blocked FVIII binding to vWF and SP fragment III. FVIII also bound to vWF which had been adsorbed to polystyrene beads. This binding was inhibited in a dose dependent manner by whole vWF, SP fragment III, and by monoclonal antibody C3. Binding could not be inhibited by SP fragment I, which contains the middle portion of the vWF molecule, or by reduced and alkylated whole vWF. SP fragment II caused only minor inhibition. Trypsin cleavage of SP fragment III produced a 35-kDa fragment containing the amino-terminal 272 amino acid residues of vWF. This fragment reacted with monoclonal antibody C3 and inhibited the binding of FVIII to vWF in a dose dependent manner. The other major fragment obtained from this digestion was a two chain hetero-dimer composed of amino acid residues 273-511 and 674-728. This fragment did not inhibit FVIII binding. These studies demonstrate that a major FVIII binding site resides within the first 272 amino acid residues of vWF.

In plasma, von Willebrand factor (vWf) associates with Factor VIII (FVIII); however, the site at which these proteins first interact has not been defined. Administration of 1-desamino-8-D-arginine vasopressin (DDAVP) causes a rapid, concomitant elevation in plasma levels of both vWf and FVIII, suggesting the existence of a DDAVP-releasable storage pool for both proteins. To determine whether vWf and FVIII can associate intracellularly and colocalize to storage vesicles, we transfected AtT-20 cells with vWf and FVIII expression plasmids. FVIII alone was not detectable within storage granules; however, transfection of vWf cDNA into the same cell caused FVIII to alter its intracellular trafficking and to undergo granular storage, colocalizing to the vWf-containing granules. In contrast, colocalization of FVIII was not observed when these cells were transfected with plasmids encoding defective FVIII-binding vWf mutants. Transfection of bovine endothelial cells with FVIII further demonstrated vesicular storage of FVIII with vWf in Weibel-Palade bodies. Since gene therapy of hemophilia A may ultimately target endothelium or hematopoietic stem cells, the interaction between vWf and FVIII within a secretory cell is important. Thus, vWf can alter the intracellular trafficking of FVIII from a constitutive to a regulated secretory pathway, thereby producing an intracellular storage pool of both proteins.

Oestradiol (E2) stimulates the growth of hormone-dependent breast cancer. 17b-hydroxysteroid dehydrogenases (17b-HSDs) catalyse the pre-receptor activation/inactivation of hormones and other substrates. 17b-HSD1 converts oestrone (E1) to active E2, but it has recently been suggested that another 17b-HSD, 17b-HSD12, may be the major enzyme that catalyses this reaction in women. Here we demonstrate that it is 17b-HSD1 which is important for E2 production and report the inhibition of E1-stimulated breast tumor growth by STX1040, a non-oestrogenic selective inhibitor of 17b-HSD1, using a novel murine model. 17b-HSD1 and 17b-HSD12 mRNA and protein expression, and E2 production, were assayed in wild type breast cancer cell lines and in cells after siRNA and cDNA transfection. Although 17b-HSD12 was highly expressed in breast cancer cell lines, only 17b-HSD1 efficiently catalysed E2 formation. The effect of STX1040 on the proliferation of E1-stimulated T47D breast cancer cells was determined in vitro and in vivo. Cells inoculated into ovariectomised nude mice were stimulated using 0.05 or 0.1 lg E1 (s.c.) daily, and on day 35 the mice were dosed additionally with 20 mg/kg STX1040 s.c. daily for 28 days. STX1040 inhibited E1-stimulated proliferation of T47D cells in vitro and significantly decreased tumor volumes and plasma E2 levels in vivo. In conclusion, a model was developed to study the inhibition of the major oestrogenic 17b-HSD, 17b-HSD1, in breast cancer. Both E2 production and tumor growth were inhibited by STX1040, suggesting that 17b-HSD1 inhibitors such as STX1040 may provide a novel treatment for hormone-dependent breast cancer.

The effective activation of factor X by factor IXa requires the co- factor activity of activated factor VIII (FVIII). Factor Xa formation is also dependent on the presence of negatively charged phospholipid. A phospholipid binding domain of FVIII has been reported to be present on the FVIII light chain. Recent observations on a subset of human FVIII inhibitors have implicated the carboxyl-terminal C2 domain of FVIII as containing a possible phospholipid binding site. The purpose of this study was to investigate directly the role of the C2 domain in phospholipid binding. Twenty-six overlapping peptides, which span the entire C2 domain of FVIII, were synthesized. The ability of these peptides to inhibit the binding of purified human FVIII to immobilized phosphatidylserine was evaluated in an enzyme-linked immunosorbent assay. Three overlapping synthetic FVIII peptides, 2303–2317, 2305- 2332, and 2308–2322, inhibited FVIII binding to phosphatidylserine by greater than 90% when tested at a concentration of 100 mumols/L. A fourth partially overlapping peptide, 2318–2332, inhibited FVIII binding by 65%. These results suggest that the area described by these peptides, residues 2303 to 2332, may play an important role in the mediation of FVIII binding to phospholipid.

SummaryAn international registry was established on the reproductive health of women with types of von Willebrand disease (vWd) unresponsive to DDAVP. Data was collected on 44 women from 16 treatment centers in nine countries. Severe menorrhagia requiring blood product therapy occurred at least once in 80% of the women for whom data was reported. Most of the reported episodes occurred prior to the diagnosis of vWd and/or the use of oral contraceptive (OC) therapy. OC therapy was clinically effective in the treatment of chronic menorrhagia in 22 of 25 (88%) women treated. Two of the women, however, were unable to tolerate chronic OC use and a third became refractory to treatment. Hysterectomy was performed in 10 of the 44 women (23%). The reported indication for six of the procedures was menorrhagia. Seventy percent were performed at two treatment centers, suggesting different thresholds for the performance of the procedure. There were 69 pregnancies reported in 31 of the 44 women. Fifteen of the pregnancies resulted in spontaneous abortions. The incidence of miscarriage was 22% and appeared clustered, with 10 of 15 of the miscarriages occurring in just four of the women

Estrogens and androgens are instrumental in the maturation of many hormone-dependent cancers. Consequently, the enzymes involved in their synthesis are cancer therapy targets. One such enzyme, steroid sulfatase (STS), hydrolyses estrone sulfate, and dehydroepiandrosterone sulfate to estrone and dehydroepiandrosterone respectively. These are the precursors to the formation of biologically active estradiol and androstenediol. This review focuses on three aspects of STS inhibitors: 1) chemical development, 2) biological activity, and 3) clinical trials. The aim is to discuss the importance of estrogens and androgens in many cancers, the developmental history of STS inhibitor synthesis, the potency of these compounds in vitro and in vivo and where we currently stand in regards to clinical trials for these drugs. STS inhibitors are likely to play an important future role in the treatment of hormone-dependent cancers. Novel in vivo models have been developed that allow pre-clinical testing of inhibitors and the identification of lead clinical candidates. Phase I/II clinical trials in postmenopausal women with breast cancer have been completed and other trials in patients with hormone-dependent prostate and endometrial cancer are currently active. Potent STS inhibitors should become therapeutically valuable in hormone-dependent cancers and other non-oncological conditions.