Biomedical subjects

P Vihko

Publications and source records attributed to P Vihko.

At least 19 recordsLinked to original sources

PubMed2004

Sex steroid hormone metabolism and prostate cancer.

The growth and function of the prostate is dependent on androgens. The two predominant androgens are testosterone, which is formed in the testis from androstenedione and 5alpha-dihydrotestosterone, which is formed from testosterone by 5alpha-reductases and is the most active androgen in the prostate. Prostate cancer is one of the most common cancers among men and androgens are involved in controlling the growth of androgen-sensitive malignant prostatic cells. The endocrine therapy used to treat prostate cancer aims to eliminate androgenic activity from the prostatic tissue. Most prostate cancers are initially responsive to androgen withdrawal but become later refractory to the therapy and begin to grow androgen-independently. Using LNCaP prostate cancer cell line we have developed a cell model to study the progression of prostate cancer. In the model androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in the oxidative 17beta-hydroxysteroid dehydrogenase activity was seen whereas the reductive activity seemed to increase. The changes suggest that during transformation estrogen influence is increasing in the cells. This is supported by the cDNA microarray screening results which showed over-expression of several genes up-regulated by estrogens in the LNCaP cells line representing progressive prostate cancer. Since local steroid metabolism controls the bioavailability of active steroid hormones in the prostate, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of the organ.

17-Hydroxysteroid Dehydrogenases↗

PubMed2004

17 beta-hydroxysteroid dehydrogenases--their role in pathophysiology.

17 beta-Hydroxysteroid dehydrogenases (17HSDs) regulate the biological activity of sex steroid hormones in a variety of tissues by catalyzing the interconversions between highly active steroid hormones, e.g. estradiol and testosterone, and corresponding less active hormones, estrone and androstenedione. Epidemiological and endocrine evidence indicates that estrogens play a role in the etiology of breast cancer, while androgens are involved in mechanisms controlling the growth of normal and malignant prostatic cells. Using LNCaP prostate cancer cell lines, we have developed a cell model to study the progression of prostate cancer. In the model LNCaP cells are transformed in culture condition into more aggressive cells. Our data suggest that substantial changes in androgen and estrogen metabolism occur in the cells, leading to increased production of active estrogens during the process. In breast cancer, the reductive 17HSD type 1 activity is predominant in malignant cells, while the oxidative 17HSD type 2 mainly seems to be present in non-malignant breast epithelial cells. Deprivation of an estrogen response by using specific 17HSD type 1 inhibitors is a tempting approach in treating estrogen-dependent breast cancer. Our recent studies demonstrate that in addition to sex hormone target tissues, estrogens may be important in the development of cancer in some other tissues previously not considered to be estrogen target tissues, such as the gastrointestinal tract.

17-Hydroxysteroid Dehydrogenases↗

PubMed2002

17 beta-hydroxysteroid dehydrogenases and cancers.

17 beta-Hydroxysteroid dehydrogenases (17HSDs) catalyze the interconversions between active 17 beta-hydroxysteroids and less-active 17-ketosteroids thereby affecting the availability of biologically active estrogens and androgens in a variety of tissues. The enzymes have different enzymatic properties and characteristic cell-specific expression patterns, suggesting differential physiological functions for the enzymes. Epidemiological and endocrine evidence indicate that estrogens play a key role in the etiology of breast cancer while androgens are involved in mechanisms controlling the growth of prostatic cells, both normal and malignant. Recently, we have developed, using LNCaP prostate cancer cell lines, a cell model to study the progression of prostate cancer. In the model LNCaP cells are transformed in culture condition to more aggressive cells, able to grow in suspension cultures. Our results suggest that substantial changes in androgen and estrogen metabolism occur in the cells during the process. These changes lead to increased production of active estrogens during transformation of the cells. Data from studies of breast cell lines and tissues suggest that the oxidative 17HSD type 2 may predominate in human non-malignant breast epithelial cells, while the reductive 17HSD type 1 activity prevails in malignant cells. Deprivation of an estrogen response by using specific 17HSD type 1 inhibitors is a tempting approach to treat estrogen-dependent breast cancer. Our recent studies demonstrate that in addition to sex hormone target tissues, estrogens may be important in the development of cancer in some other tissues previously not considered as estrogen target tissues such as colon. Our data show that the abundant expression of 17HSD type 2 present in normal colonic mucosa is significantly decreased during colon cancer development.

17-Hydroxysteroid Dehydrogenases↗

PubMed2002

Retinoic acids promote the action of aromatase and 17beta-hydroxysteroid dehydrogenase type 1 on the biosynthesis of 17beta-estradiol in placental cells.

The biosynthesis of 17beta-estradiol (E(2)) in human placenta involves the actions of aromatase and 17beta-hydroxysteroid dehydrogenase type 1 (17HSD1). Aromatase, an enzyme complex comprised of P450aromatase (P450arom) and NADH-cytochrome P450 reductase, converts androgens to estrogens, whereas 17HSD1 catalyzes the reduction of estrone to E(2). In the present study, the effects of retinoic acids (RAs) on P450arom and 17HSD1 expression in placental cells were investigated. Treatment with all-trans-RA (at-RA) or 9cis-RA increased E(2) production in JEG-3 choriocarcinoma cells and cytotrophoblast (CTB) cells isolated from normal early placentas. Meanwhile, the activity of aromatase and expression of P450arom mRNA were induced by at-RA in JEG-3 cells. Northern blot analysis showed that the effect on P450arom mRNA expression occurs in a dose- and time-dependent fashion. Similar to at-RA and 9cis-RA, Ro40-6055, the retinoic acid receptor alpha (RARalpha)-selective activator, increased the expression of P450arom and 17HSD1 mRNA in JEG-3 cells. On the other hand, Ro41-5253 (Ro41), the RARalpha-selective antagonist, blocked the stimulatory effect of RAs on P450arom expression. Surprisingly, Ro41 induced the activity and mRNA expression of 17HSD1 in JEG-3 cells, which is in contrast to the expected inhibitory effect and, moreover, remarkably potentiated the induction by at-RA and 9cis-RA. However, reporter gene analysis revealed that the influence of Ro41 on the transcription of the HSD17B1 gene, which encodes 17HSD1, is considerably milder in JEG-3 cells, and it only additively enhanced the effect of at-RA. Finally, it was found that at-RA and 9cis-RA increased the expression of P450arom and 17HSD1 mRNA in CTB cells, but to a lesser extent. The data suggest that RAs may play a role in promoting the biosynthesis of E(2 )in the placenta. In addition, Ro41 has divergent effects on gene expression in JEG-3 cells.

17-Hydroxysteroid Dehydrogenases↗

PubMed2001

The TMPRSS2 gene encoding transmembrane serine protease is overexpressed in a majority of prostate cancer patients: detection of mutated TMPRSS2 form in a case of aggressive disease.

The serine protease TMPRSS2 gene expression was studied by in situ hybridization using benign prostatic hyperplasia and prostate cancer tissue samples from 32 patients. Expression of TMPRSS2 gene was higher in cancer cells than that in benign cells in 84% of the specimens containing both benign and malignant tissues. The TMPRSS2 mRNA level was significantly increased in poorly differentiated (p = 0.014, n = 7) and untreated (p = 0.022, n = 13) primary prostate adenocarcinomas compared to benign tissues. In addition, androgen-deprivation therapy significantly decreased the expression of TMPRSS2 in benign prostate tissue (p = 0.07), which is in accordance with the androgen-inducible expression of the gene. The gene copy number of TMPRSS2, analyzed by competitively differential PCR, was duplicated in the malignant cells of about 38% of the prostate cancer patients analyzed. Thus, the increase in the gene copy number is probably not the primary reason for the detected overexpression of the TMPRSS2 gene. Mutations in the TMPRSS2 gene were screened using DNA isolated from paraffin-embedded prostate cancer tissues from 9 patients with aggressive prostate cancer and from 9 patients with nonaggressive disease. Thirteen exons covering the coding region were checked using enzymatic mutation detection and direct sequencing. One patient with aggressive prostate cancer carried a deletion and a stop codon in exon 11, leading to inactivation of the serine protease domain in TMPRSS2.

Humans↗

PubMed2001

Specific DNA binding and transactivation potential of recombinant, purified Stat5.

The signal transducers and activators of transcriptions (Stats) are central mediators of cytokine responses especially in hematopoietic cells. The detailed molecular mechanisms of Stat activation, particularly the role of post-translational modifications and co-operation with cellular transcription factors are subject to intense investigation. The phosphorylation of a tyrosine residue in the carboxyl terminal domain is a common characteristic for the biologically active state of all known Stats. We studied the biological potential of purified recombinant murine Stat5a and Stat5b. These proteins were expressed in Sf9 insect cells upon infection with Stat5 encoding baculoviruses. We also obtained the tyrosine phosphorylated, activated forms of the Stat5 proteins by expressing the tyrosine kinase Janus kinase2 (Jak) in the same cells through co-infection with a kinase encoding virus. After purification, only the tyrosine phosphorylated form was able to bind specifically in vitro to the Stat5 DNA response element. This activated form of Stat5 is also able to support specific cell free in vitro transcription of a gene with a Stat5 response element in its promoter region. The recombinant purified Stat5 proteins were treated with the tyrosine specific protein phosphatase or with potato acidic phosphatase, which removes phosphate groups from serine and tyrosine residues. Phosphatase treatment resulted in the loss of specific DNA binding ability. This property could be restored by an in vitro reaction with recombinant, purified EGF or PDGF receptor kinases. Tyrosine rephosphorylation in vitro also restored the transactivation potential of Stat5. This modification is, therefore, a sufficient prerequisite for transcriptional induction by Stat5.

Animals↗

PubMed2001

Ligand affinity, homodimerization, and ligand-induced secondary structural change of the human vitamin d receptor.

The intrinsic tryptophan fluorescence signal of the full-length nuclear receptor hVDR was used to directly determine the dissociation constants, K(d), of two ligands yielding K(d) = 32 nM for 1alpha,25(OH)(2)D(3) and K(d) = 322 nM for 25(OH)D(3). Ligand binding was accompanied by a conformational change in the alpha-helical part of hVDR as revealed by CD spectroscopy. In addition, the presence of calcitriol was found to be a necessary prerequisite for the homodimerisation of hVDR which was monitored using fluorescence anisotropy. We conclude that the observed ligand-induced structural change of hVDR is conditional for dimerisation of the protein.

Calcifediol↗

PubMed2001

Cytokine regulation of the expression of estrogenic biosynthetic enzymes in cultured rat granulosa cells.

Both P450 aromatase (P450arom) and 17beta-hydroxysteroid dehydrogenase (17HSD) type 1 are key enzymes in the ovarian E(2) biosynthesis. Cytokines have been suggested to be mediators between the immune and the reproductive systems, and they may play a role as paracrine or autocrine ovarian regulatory factors. Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha) have been shown to modulate the FSH-induced E(2) production in immature rat granulosa cells. The aim of the present study was to investigate the effects of these cytokines on the activity and expression of the 17HSD type 1 enzyme in cultured undifferentiated granulosa cells. Furthermore, the expression of P450arom was also analyzed. The granulosa cells obtained from the ovaries of immature DES-treated rats were initially cultured for 48 h with no other treatment and then incubated with or without the test reagents for an additional 48 h. The treatment of the granulosa cells with cytokines alone did not affect the activity of 17HSD type 1 as assessed by the conversion of tritiated substrate. However, both TNFalpha and IL-1beta caused a dose-dependent inhibition of the recombinant FSH-induced enzyme activity and the Forskoline-induced expression of 17HSD type 1 and P450arom mRNAs. The cytokines only slightly inhibited the 8-Br-cAMP-induced P450arom expression. In contrast, the inhibitory cytokine effects on 17HSD type 1 expression and activity were not abolished by the presence of 8-Br-cAMP. Despite the presence of inhibitors of protein kinase C (staurosporine) or tyrosine kinases (genistein), the inhibitory effects of TNFalpha and IL-1beta on the Forskoline-induced expression of 17HSD type 1 and P450arom and the Forskoline-induced 17HSD activity were not blocked. The data show a dose dependent inhibitory effect of TNFalpha and IL-1beta on gonadotropin action, opposite to the follicular development by down-regulating the expressions of estrogen biosynthetic enzymes. The cytokine effects on P450arom expression are mainly derived from a decrease in gonadotropin-induced cAMP production, while the inhibitory mechanisms on 17HSD type 1 expression involve distal sites from cAMP generation. The protein kinase C and tyrosine kinase pathways are likely not to be involved in the latter mechanisms.

17-Hydroxysteroid Dehydrogenases↗

PubMed2001

Molecular mechanisms of estrogen recognition and 17-keto reduction by human 17beta-hydroxysteroid dehydrogenase 1.

The reduction of inactive estrone (E1) to the active estrogen 17beta-estradiol (E2) is catalyzed by type 1 17beta-hydroxysteroid dehydrogenase (17HSD1). Crystallographic studies, modeling and activity measurement of mutants and chimeric enzymes have led to the understanding of its mechanism of action and the molecular basis for the estrogenic specificity. An electrophilic attack on the C17-keto oxygen by the Tyr 155 hydroxyl is proposed for initiation of the transition state. The active site is a hydrophobic pocket with catalytic residues at one end and the recognition machinery on the other. Tyr 155, Lys 159 and Ser 142 are essential for the activity. The presence of certain other amino acids near the substrate recognition end of the active site including His 152 and Pro 187 is critical to the shape complementarity of estrogenic ligands. His 221 and Glu 282 form hydrogen bonds with 3-hydroxyl of the aromatic A-ring of the ligand. This mechanism of recognition of E1 by 17HSD1 is similar to that of E2 by estrogen receptor alpha. In a ternary complex with NADP(+) and equilin, an equine estrogen with C7=C8 double bond, the orientation of C17=O of equilin relative to the C4-hydride is more acute than the near normal approach of the hydride for the substrate. In the apo-enzyme structure, a substrate-entry loop (residues 186-201) is in an open conformation. The loop is closed in this complex and Phe 192 and Met 193 make contacts with the ligand. Residues of the entry loop could be partially responsible for the estrogenic specificity.

17-Hydroxysteroid Dehydrogenases↗

PubMed2001

Structure and function of 17beta-hydroxysteroid dehydrogenase type 1 and type 2.

17beta-Hydroxysteroid dehydrogenases (17HSDs) catalyze the interconversions between high-activity 17beta-hydroxysteroids and low-activity 17-ketosteroids. Several distinct 17HSD isoenzymes have been characterized. They have unique tissue distribution patterns suggesting a specific function for each of the isoenzymes in modifying sex steroid hormone activity. The activities of 17HSDs are essential for gonadal sex steroid biosynthesis and they are also involved in the modulation of steroid hormone action in peripheral tissues. 17HSD type 1 (17HSD1) is needed for estradiol biosynthesis in ovarian granulosa cells and it is also expressed in breast tissue, thus increasing locally estradiol concentration. 17HSD type 2 (17HSD2) is another 17HSD enzyme involved in estrogen metabolism. The type 2 enzyme has an opposite activity catalyzing estradiol to estrone, thereby reducing the exposure of tissues to estrogen action. Preliminary data suggest that 17HSD2 may predominate in human non-malignant breast epithelial cells, while 17HSD type 1 activity prevails in malignant cells. Determination of the three-dimensional structure of human 17HSD1 has led to an atomic level description of the estradiol binding pocket of the enzyme and an understanding of its mechanism of action, and the molecular basis for the estrogen-specificity of the enzyme. Deprivation of an estrogen response by using specific 17HSD1 inhibitors is a tempting approach to treat estrogen-dependent breast cancer.

17-Hydroxysteroid Dehydrogenases↗

PubMed2001

Colorectal carcinoma associated with serrated adenoma--prevalence, histological features, and prognosis.

Serrated adenoma has been proposed to be a distinct entity among colorectal neoplasms. Progression to frank carcinoma has been suggested in individual cases, but the prevalence of carcinomas originating from serrated adenomas and their clinico-pathological characteristics are not known. In the present study, a large series of colorectal cancers was analysed for the occurrence of serrated adenoma in association with carcinoma and clinico-pathological features were compared in cases with and without serrated adenoma. Specimens from 466 colorectal carcinoma patients undergoing operations between 1986 and 1996 were re-evaluated for the presence of juxtaposed serrated adenoma and carcinoma. Clinico-pathological features such as location, Dukes' stage, histological grade, mucinous differentiation, and prognosis were evaluated. Twenty-seven carcinomas (5.8%) were found in association with an adjacent serrated adenoma. Eight of the patients were male and 19 were female. All of these adenocarcinomas showed a serrated appearance resembling that of serrated adenomas. Nine (33%) cases were mucinous and a mucinous component was present in 11 (41%) additional cases. The majority of the tumours were located either in the caecum (14 cases; 51%) or in the rectum (9 cases; 33%). DNA microsatellite instability was more common in carcinomas associated with serrated adenoma (37.5%) than in other carcinomas (11.0%). It is concluded that carcinoma associated with serrated adenoma is a distinct type of colorectal neoplasm, accounting for 5.8% of all colorectal carcinoma cases in this study. Predilection for the caecum and the rectum may reflect their aetiological factors. Female preponderance is contrary to that reported for hyperplastic polyps and serrated adenomas.

Adenocarcinoma↗

PubMed2001

Functional and structural characterization of the insertion region in the ligand binding domain of the vitamin D nuclear receptor.

Vitamin D nuclear receptor mediates the genomic actions of the active form of vitamin D, 1,25(OH)2D3. This hormone is involved in calcium and phosphate metabolism and cell differentiation. Compared to other nuclear receptors, VDR presents a large insertion region at the N-terminal part of the ligand binding domain between helices H1 and H3, encoded by an additional exon. This region is poorly conserved in VDR in different species and is not well ordered as observed by secondary structure prediction. We engineered a VDR ligand binding domain mutant by removing this insertion region. Here we report its biochemical and biophysical characterization. The mutant protein exhibits the same ligand binding, dimerization with retinoid X receptor and transactivation properties as the wild-type VDR, suggesting that the insertion region does not affect these main functions. Solution studies by small angle X-ray scattering shows that the conformation in solution of the VDR mutant is similar to that observed in the crystal and that the insertion region in the VDR wild-type is not well ordered.

Amino Acid Sequence↗

PubMed2000

Classification of advanced colorectal carcinomas by tumor edge morphology: evidence for different pathogenesis and significance of polypoid and nonpolypoid tumors.

BACKGROUND: Increasing evidence suggests that a substantial proportion of colorectal carcinomas develop without a preexisting polypoid adenomatous lesion, but it is difficult to detect the possible origin of advanced carcinomas. The purpose of this study was to test the validity and significance of a new histopathologic classification system based on the histologic analysis of the tumor edge. METHODS: One hundred eighty-six unselected cases of colorectal carcinoma were included. A new classification method to distinguish polypoid and nonpolypoid growth type was based on the presence or absence of elevation of tumor as compared with adjacent mucosa. Inter- and intraobserver agreement of classification was tested. Association with other clinicopathologic features including histopathologic characteristics of the tumors, presence or absence of lesional and concurrent adenoma, K-ras mutations, and prognosis was evaluated. RESULTS: Classification could be made in 75% of the tumors, and 25% were unclassifiable, mostly due to absence of tumor margin in sections. Of the classifiable carcinomas, 45% were classified as polypoid, of which 52% had lesional adenoma. Nonpolypoid tumors formed 48% of classifiable cases, and only 2% had lesional adenoma. Features of both polypoid and nonpolypoid carcinomas were present in 7% of cases. Concurrent extralesional adenomas were found more frequently in association with polypoid carcinomas. K-ras mutations were more common in polypoid (43%) than in nonpolypoid tumors (8%; P = 0.018). Nonpolypoid carcinomas were significantly (P = 0.03) more aggressive than polypoid carcinoma, with 38% and 20% recurrence rates, respectively. CONCLUSIONS: The authors' results indicate that advanced colorectal carcinomas can be classified according to growth pattern by observing the tumor edge. This classification has prognostic significance because nonpolypoid carcinomas appeared to have a worse prognosis than polypoid ones.

Adenoma↗

PubMed2000

Estrogen metabolism as a regulator of estrogen action in the mammary gland.

Estrogen action in the target cells is dependent on estrogen receptor activity and intracellular estrogen concentration, which, in turn, is affected by the serum concentration and local metabolism in these cells. During the reproductive years the main source of estrogens is the ovarian follicles, but in postmenopausal women most of the estrogens are formed in peripheral tissues. 17Beta-hydroxysteroid dehydrogenases (17HSDs) catalyze the reaction between 17beta-hydroxysteroids and 17-ketosteroids, and several distinct 17HSD isoenzymes have been characterized. 17HSD type 1 catalyzes the reaction from low-activity estrone to high-activity estradiol. The type 2 enzyme has an opposite activity, thereby reducing the exposure of tissues to estrogen action. 17HSD type 1 is expressed both in steroidogenic tissues and in the target tissues of steroid action, such as normal and malignant breast tissue, where it may be responsible for maintaining the high intracellular estradiol concentration seen in breast cancer specimens. Therefore, 17HSD type 1 inhibitors may be useful in the treatment and/or prevention of estrogen-dependent malignancies, such as breast cancer. This article deals mainly with 17HSD types 1 and 2 and their role in estrogen action in breast tissue.

17-Hydroxysteroid Dehydrogenases↗

PubMed2000

Differentially expressed genes in two LNCaP prostate cancer cell lines reflecting changes during prostate cancer progression.

Prostate cancer tends to become transformed to androgen-independent disease over time when treated by androgen-deprivation therapy. We used two variants of the human prostate cancer cell line LNCaP to study gene expression differences during prostate cancer progression to androgen-independent disease. Production of prostate-specific antigen was regarded as a marker of androgen-dependence and loss of prostate-specific antigen was regarded as a marker of androgen-independence. mRNA from both cell lines was used for cDNA microarray screening. Differential expression of several genes was confirmed by Northern blotting. Monoamine oxidase A, an Expressed Sequence Tag (EST) similar to rat P044, and EST AA412049 were highly overexpressed in androgen-dependent LNCaP cells. Tissue-type plasminogen activator, interferon-inducible protein p78 (MxB), an EST similar to galectin-1, follistatin, fatty acid-binding protein 5, EST AA609749, annexin I, the interferon-inducible gene 1-8U, and phospholipase D1 were highly overexpressed in androgen-independent LNCaP cells. All studied genes had low or no expression in PC-3 cells. The EST similar to rat P044, the EST similar to galectin-1, follistatin, annexin I, and the interferon-inducible gene 1-8U were also expressed in benign prostatic hyperplasia tissue. The Y-linked ribosomal protein S4, Mat-8, and EST AA307912 were highly expressed in benign prostatic hyperplasia tissue. Additionally, both confirmation of differential expression in Northern blots and in situ hybridization were carried out for monoamine oxidase A, the EST similar to rat P044, the EST similar to galectin-1, fatty acid-binding protein 5, and the interferon-inducible gene 1-8U. We identified several potential prostate cancer markers, indicating that the method used is a useful tool for the screening of cancer markers, but other methods, such as in situ hybridization, are needed to further investigate the observations.

Animals↗

PubMed2000

Expression of mouse 17beta-hydroxysteroid dehydrogenase/17-ketosteroid reductase type 7 in the ovary, uterus, and placenta: localization from implantation to late pregnancy.

Rodent 17beta-hydroxysteroid dehydrogenase/17-ketosteroid reductase type 7 (17HSD/KSR7) catalyzes the conversion of estrone (E1) to estradiol (E2) and is abundantly expressed in the ovaries of pregnant animals in particular. In the present work we demonstrate cell-specific expression of 17HSD/KSR7 in the ovaries, uteri, and placentas of pregnant and nonpregnant mice using in situ hybridization. The results show that mouse 17HSD/KSR7 (m17HSD/KSR7) messenger RNA is distinctly and exclusively expressed in a proportion of corpora lutea (CLs). During pregnancy, expression of m17HSD/KSR7 is most abundant around embryonic day 14.5 (E14.5), when the ovaries are filled with CLs expressing 17HSD/KSR7. In the uterus, m17HSD/KSR7 is first detected on E5.5, when expression surrounds the implantation site on the antimesometrial side. As gestation progresses, m17HSD/KSR7 is expressed in the decidua capsularis on E8 and E9.5, disappearing thereafter from the antimesometrial decidua. On E9 onward, m17HSD/KSR7 messenger RNA expression takes place at the junctional zone of the developing placenta. On E12.5 and E14.5, m17HSD/KSR7 is abundantly expressed in the spongiotrophoblasts, where expression gradually declines toward parturition. In conclusion, m17HSD/KSR7 expression in the CL is related to the life span of the CL. Moreover, spatial and temporal expression of m17HSD/KSR7 in the uterus suggests that locally produced E2 plays a role in implantation and/or decidualization. Finally, the results indicate that mouse placenta is capable of converting E1 to E2 in situ, and that the synthesized E2 may be effective in a paracrine, autocrine, and/or intracrine manner and be involved in placentation.

17-Hydroxysteroid Dehydrogenases↗

PubMed1999

Large-scale expression and purification of the human vitamin D receptor and its ligand-binding domain for structural studies.

We have expressed recombinant human vitamin D receptor and its ligand-binding domain in Spodoptera frugiperda (Sf9) insect cells with a 30-litre bioreactor. Both proteins were purified to apparent homogeneity with yields of 0.5-3.5 mg/l. Gel-filtration analyses indicated that the purified human vitamin D receptor and its ligand-binding domain were present as monomers in solution. The purified vitamin D receptor and its ligand-binding domain were demonstrated to bind 1alpha,25-dihydroxyvitamin D(3) with high affinity, the K(d) values ranging from 0.9 to 1.2 nM. Neutron scattering studies of the ligand-binding domain demonstrated that the samples are homogeneous and contain monomeric species of polypeptides. The purified vitamin D receptor binds to the vitamin D response elements of osteopontin and osteocalcin genes as a homodimer or as a heterodimer with the retinoid X receptor-alphaDeltaAB and we were able to purify these complexes in quantities sufficient for crystallization studies. The results indicate that we can produce biologically active human vitamin D receptor and its ligand-binding domain in insect cells and purify them for functional and structural studies.

Animals↗

PubMed1999

Intracellular fragmentation of bone resorption products by reactive oxygen species generated by osteoclastic tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAP) is highly expressed in bone-resorbing osteoclasts and activated macrophages. It has been suggested that a redox-active iron in the binuclear iron center of TRAP could have the capacity to react with hydrogen peroxide to produce highly destructive reactive oxygen species (ROS). Here we show that TRAP can generate ROS in vitro and that cells over-expressing TRAP produce higher amounts of intracellular ROS than their parent cells. We further demonstrate that these ROS can be targeted to destroy collagen and other proteins. In resorbing osteoclasts, TRAP was found in transcytotic vesicles transporting matrix degradation products through the cell, suggesting that TRAP-facilitated fragmentation of endocytosed material takes place in a specific cellular compartment. These results suggest that bone matrix degradation occurs not only extracellularly in the resorption lacunae but also intracellularly in the transcytotic vesicles. We propose that proteins containing redox-active iron could represent a novel mechanism of physiological fragmentation of organic molecules. This mechanism could be important in tissue remodeling and as a defense mechanism of phagocytosing cells.

Acid Phosphatase↗