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B S Komm

Publications and source records attributed to B S Komm.

At least 37 records · Page 2Linked to original sources
PubMed1992

Evaluation of a putative vitamin D response element in the avian calcium binding protein gene.

A primary response of the avian intestine to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is increased synthesis of a 28-kD calcium-binding protein, calbindin-D28k (CaBP). This study examined whether 1,25-(OH)2D3 regulates CaBP gene transcription by an interaction of the vitamin D receptor (VDR) with a vitamin D-responsive element (VDRE) in the CaBP promoter. A genomic clone of CaBP containing about 1 kb of 5'-flanking DNA and 13 kb of the structural gene was isolated. 5'-Flanking DNA from -320 to -306 had considerable sequence similarity to VDREs identified in other genes. Indeed, a portion of the CaBP gene containing this region (-743 to +47) linked to a growth hormone reporter construct elicited a 1,25-(OH)2D3-dependent, VDR-dependent increase in reporter expression in transiently transfected chicken embryo fibroblasts. However, deletion analysis demonstrated that the sequences responsible for this induction reside 3' to -133 and the putative VDRE at -320 to -306 was not involved in the response. Furthermore, transfection of heterologous promoter constructs consisting of a Ban I fragment (-354 to -252) linked to the Herpes simplex thymidine kinase promoter revealed no effect of this region on reporter expression. Gel mobility shift analysis confirmed that this putative VDRE in the CaBP promoter was not a high-affinity binding site for VDR. Consequently, functional significance with respect to the primary induction of CaBP by 1,25-(OH)2D3 cannot be ascribed to this region of the CaBP promoter.

Animals↗
PubMed1992

Stable expression of the calbindin-D28K complementary DNA interferes with the apoptotic pathway in lymphocytes.

The WEHI7.2 thymoma cell line undergoes apoptotic cell death when exposed to glucocorticoids and agents that increase intracellular cAMP. Several lines of evidence indicate that calcium may play an important role in events culminating in lymphocyte apoptosis. In these studies, calbindin-D28K was stably overexpressed in WEHI7.2 cells to determine if increasing the Ca(2+)-binding capacity of the cell interferes with the apoptotic pathway. Indeed, stable expression of calbindin-D28K decreased the apoptotic effects of dexamethasone and forskolin, and the level of resistance to these agents correlated with the relative amount of calbindin expressed in each line. Overexpression of calbindin also increased cell survival in the presence of the calcium ionophore A23187. The stably expressed calcium-binding protein appeared to exert its protective effect subsequent to transcriptional activation, since glucocorticoid- and cAMP-induced gene expression were not affected. These data support the proposal that calcium fluxes are involved in apoptosis and suggest that high level expression of proteins that buffer calcium fluxes can effectively suppress death in apoptosis-susceptible cells.

Animals↗
PubMed1991

Evidence for early induction of calmodulin gene expression in lymphocytes undergoing glucocorticoid-mediated apoptosis.

Glucocorticoid treatment of certain lymphoma cell lines and thymocytes activates a self-destructive pathway of programmed cell death referred to as apoptosis. Calcium and calmodulin (CaM) may be important signals in the apoptotic cascade because an early event is a sustained elevation in cytosolic Ca2+ and CaM inhibitors interfere with the death pathway. In the present study, expression of the CaM gene was examined during glucocorticoid-induced apoptosis in WEHI7.2 lymphocytes. Steady state levels of CaM mRNA were increased up to 10-fold following a 4-6-h exposure of WEHI7.2 cells to 10(-6) M dexamethasone. This increase was mediated through the glucocorticoid receptor since the response was not observed in WEHI7.418, a variant line which does not express active glucocorticoid receptor. Induction of CaM mRNA was dose-dependent and highly specific for glucocorticoids, as other steroids were unable to elicit the response. A stringent cell specificity was also observed. Pretreatment of WEHI7.2 lymphocytes with cycloheximide did not interfere with dexamethasone-dependent increases in CaM mRNA levels, and studies with actinomycin D demonstrated that the stability of the transcript was not altered by hormone, Finally, a calmodulin inhibitor elicited a protective effect on WEHI7.2 cells following glucocorticoid exposure. These results indicate that CaM mRNA levels were hormonally controlled in WEHI7.2 lymphocytes and support the putative involvement of CaM in glucocorticoid-induced apoptosis.

Animals↗
PubMed1991

Estrogen binding and estrogenic responses in normal human osteoblast-like cells.

A finite human cell line was established from trabecular bone explants obtained from a 48-year-old woman. These cells, designated BG688, were characterized as osteoblast-like in phenotype using the following independent criteria: (1) the presence of histochemically detectable alkaline phosphatase (AP) activity; (2) response to the calciotropic hormone 1,25-(OH)2D3 as assessed by increased AP activity; (3) synthesis and secretion of the osteoblast-specific marker bone gla protein; and (4) expression of alpha 1(I)-procollagen and alpha 1(III)-procollagen mRNAs in a pattern similar to that of other osteoblast-like cell lines. In addition to these classic osteoblast markers, BG688 cells also possess approximately 2400 high-affinity (Kd = 0.45 nM) 17 beta-estradiol (E2) binding sites per cell. The binding of E2 to these sites is specific, and of the steroid hormone agonists tested, E2 and diethylstilbestrol elicited the greatest amount of competition with radiolabeled E2. BG688 cells were also shown to respond to a physiologic concentration (10 nM) of E2. In vitro translation products of poly(A)+ RNA obtained from control and hormone-treated cells revealed a pleiotropic influence of E2 on the relative abundance of several mRNAs as assessed by two-dimensional gel electrophoretic analysis of their corresponding peptides. E2 also elicits a twofold increase in the steady-state concentration of alpha 1(I)-procollagen mRNA as demonstrated by northern blot hybridization. Thus, we here extend our previous data obtained in osteoblast-like osteosarcoma cells to indicate that a normal osteoblastic cell line is a target for the action of estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1991

High-affinity androgen binding and androgenic regulation of alpha 1(I)-procollagen and transforming growth factor-beta steady state messenger ribonucleic acid levels in human osteoblast-like osteosarcoma cells.

Clinical observations have demonstrated a positive effect of estrogens and androgens on the maintenance of structural bone integrity. This study examines the direct effects of androgenic hormones on the osteoblast-like human osteosarcoma cell line, HOS TE85. Employing radiolabeled dihydrotestosterone (DHT), 2800 saturable, high-affinity (dissociation constant = 0.66 nM) androgen binding sites were detected per HOS TE85 cell. Androgen binding was specific in that DHT and testosterone (T) displayed significantly greater competition than the progestins, progesterone and medroxyprogesterone. The expression of androgen receptors in HOS TE85 cells was further substantiated by Northern analysis. A human androgen receptor complementary DNA probe revealed a 9.5 kilobase transcript which corresponds to the predominant human androgen receptor transcript detected in human male reproductive tissues. Androgens were also found to elicit biological responses in HOS TE85 cells. Physiological concentrations of DHT and T decreased HOS TE85 cell proliferation as assessed by cell count. This finding suggests that DHT may also play a role in osteoblast differentiation. In support of this hypothesis, treatment with T (24 h, 10 nM) enhanced the abundance of both alpha 1(I)-procollagen messenger RNA (mRNA) (5-fold) and transforming growth factor-beta mRNA (2.2 fold). The nonaromatizable androgen DHT (24 h, 10 nM) elicited an increase in the steady state concentration of alpha 1(I)-procollagen mRNA similar to the increase observed with T treatment. Thus, in addition to the recent discovery of estradiol receptors and estrogenic regulation of HOS TE85 cells, it is now evident that these osteoblast-like osteosarcoma cells also express high affinity androgen binding sites and can respond biologically to androgens.

Androgens↗
PubMed1991

The vitamin D-responsive element in the rat bone Gla protein gene is an imperfect direct repeat that cooperates with other cis-elements in 1,25-dihydroxyvitamin D3- mediated transcriptional activation.

The gene for rat bone gla protein (BGP) was isolated and 1250 basepairs (bp), including 1100 bp of 5' flanking DNA, were placed up-stream of the human GH reporter gene. After transient transfection into the osteoblast-like rat osteosarcoma cell line ROS 17/2.8, the BGP promoter demonstrated a low level of basal activity that was increased approximately 10-fold by the addition of 10(-8) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. A single 250-bp fragment (-523 to -274) was sufficient to confer hormone inducibility upon both heterologous and homologous promoters. Deletion studies, complemented by evaluation with synthetic oligomers, enabled localization of the 1,25-(OH)2D3 response element to within 19 bp (-456 to -438), containing an element with an imperfect direct repeat [GGTGA(N4)GGACA] and homology to other steroid-responsive elements. Gel retardation assays demonstrated that partially purified chick intestinal 1,25-(OH)2D3 receptor bound specifically and with high affinity to a DNA fragment containing the putative 1,25-(OH)2D3 response element, and this binding was perturbed by monoclonal antibodies to the 1,25-(OH)2D3 receptor. Surprisingly, the 250-bp fragment, when linked in an antisense orientation with respect to the BGP promoter, blocked basal and hormone-dependent gene expression. However, a 246-bp fragment 5' to the 250-bp element (-1100 to -855) restored 20-fold inducibility when linked to the first fragment in the same orientation, suggesting cooperativity between at least two elements to achieve the hormonal regulation observed in this gene.

Animals↗
PubMed1990

Progesterone regulation of estradiol-induced rat uterine secretory protein, complement C3.

Previously, using DNA sequencing, Northern and Southern analysis, and immunohistochemical data, we identified an estrogen-stimulated secretory protein as the third component of complement (C3). In this study, we demonstrate that progesterone modulated the estradiol regulation of C3 in immature rats as well as during the normal reproductive cycle. C3 was most abundant during estrus and reached its lowest concentration in diestrus. Immunoprecipitations reveal that progesterone prevented the estradiol-stimulated increase in radiolabeled C3 both in the media and tissue. The mechanism for the progesterone inhibition of estrogen-stimulated C3 appeared to be at the level of transcription or possibly mRNA stability since progesterone blocked the estradiol-stimulated increase in C3 mRNA.

Animals↗
PubMed1990

The stimulation of uterine complement component C3 gene expression by antiestrogens.

We have previously demonstrated that complement component C3 is regulated by estradiol in the rat uterus. The antiestrogens tamoxifen, LY117018, and LY156758 exert both agonist and antagonist effects on the immature rat uterus. In this study, these three antiestrogens also stimulated an increase in the synthesis and secretion of C3. The combination of LY117018 and estradiol did not increase C3 to a greater extent than LY117018 alone, which suggests a similar mechanism of regulation. The regulation may be transcriptional since both estradiol and tamoxifen increase the concentration of C3 mRNA. Results of in situ hybridization revealed that the increase in C3 mRNA occurred in the luminal epithelial cells. Although the induction by estradiol and the antiestrogens was similar in most aspects, the time course for tamoxifen-stimulated synthesis differed from estradiol in that the time required to achieve maximal concentrations of C3 was delayed by 12 h with tamoxifen. This pattern did not appear to be related to the time it took to convert tamoxifen to 4-hydroxytamoxifen since the C3 response for these antiestrogens were identical. The antiestrogen-stimulated increase in C3 synthesis and mRNA concentration was prevented by the co-administration of progesterone lending support to the hypothesis that the antiestrogens regulate C3 synthesis via a mechanism similar to estrogen.

Animals↗
PubMed1989

Estrogen regulation of tissue-specific expression of complement C3.

The administration of estradiol to immature rats results in the increased synthesis and secretion of a 180-kDa protein, composed of 115- and 65-kDa subunits, by the uterine luminal epithelial cells. A monoclonal antibody against the 180-kDa protein was utilized to isolate the corresponding cDNA (LE-1) from a rat uterine luminal epithelial cell cDNA lambda gt11 expression library. This LE-1 cDNA was sequenced and shown to be homologous to complement component C3. The sequence was approximately 81 and 90% homologous to human and mouse C3, respectively. The LE-1 cDNA sequence was homologous with the 3' portion of the C3 mRNA containing the alpha subunit (115 kDa). Uterine mRNA isolated from immature rats treated with 1 microgram of estradiol for 24 h demonstrated a 25-fold increase in the concentration of a 6.0-kilobase mRNA by Northern hybridization with either LE-1 or authentic human C3 cDNA probes. To further examine the possibility that the estradiol-regulated secretory protein was C3, an aliquot of radiolabeled media protein from control and estradiol-stimulated rat uteri was incubated with goat anti-rat C3 antibody. The immunoprecipitated radiolabeled protein from estradiol-treated animals was increased significantly (p less than 0.01) compared to media from control animals. Analysis of the immunoprecipitated proteins on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of 180 kDa from estradiol-stimulated uterine media, whereas no detectable proteins were immunoprecipitated from media obtained from control uteri. Also, when the immunoprecipitated protein was reduced (20 mM dithiothreitol) it dissociated into two subunits of 115 and 65 kDa. Immunohistochemical studies demonstrated the presence of C3 only in the epithelial cells of estrogen-stimulated rat uteri. In addition, the estradiol-stimulated mRNA was only detectable in uterine epithelial cell RNA. Analysis of liver RNA demonstrated a 6.0-kilobase mRNA, as in the uterus, when hybridized with LE-1. However, unlike the uterus, its concentration was not influenced by estrogen administration with up to three daily injections of 100 micrograms of diethylstilbestrol. Based on biophysical, DNA sequence, and antibody data we conclude that rat uterine epithelial cells produce C3 in response to estradiol whereas the expression in the liver was not modulated by estrogens.

Animals↗
PubMed1988

Estrogen binding, receptor mRNA, and biologic response in osteoblast-like osteosarcoma cells.

High specific activity estradiol labeled with iodine-125 was used to detect approximately 200 saturable, high-affinity (dissociation constant approximately equal to 1.0 nM) nuclear binding sites in rat (ROS 17/2.8) and human (HOS TE85) clonal osteoblast-like osteosarcoma cells. Of the steroids tested, only testosterone exhibited significant cross-reactivity with estrogen binding. RNA blot analysis with a complementary DNA probe to the human estrogen receptor revealed putative receptor transcripts of 6 to 6.2 kilobases in both rat and human osteosarcoma cells. Type I procollagen and transforming growth factor-beta messenger RNA levels were enhanced in cultured human osteoblast-like cells treated with 1 nM estradiol. Thus, estrogen can act directly on osteoblasts by a receptor-mediated mechanism and thereby modulate the extracellular matrix and other proteins involved in the maintenance of skeletal mineralization and remodeling.

Animals↗
PubMed1988

Cloning of estrogen-regulated messenger ribonucleic acids from rat uterus.

A cDNA library prepared from the mRNA of uteri of estrogen-stimulated immature female rats was constructed in lambda gt10. Differential screening of the hybrid phages was performed using control and stimulated cDNAs as probes. Selected clones were then characterized by Northern and Southern blot hybridizations. In all, eight unique clones corresponding to estrogen-stimulated messages in rat uterus were identified. These clones hybridized to uterine mRNAs varying in size from 1.4-8.4 kilobases. Three of the clones were characterized as coding for three different types of collagen, and one as coding for smooth muscle actin. These are described in more detail elsewhere. The kinetics of increase in estrogen-regulated messages was examined. After a single injection of estradiol, five clones, including the three collagen mRNAs, showed two peaks of accumulation, at 4 and 24 h. Messages of two other clones were maximal at 12 h. The actin clone hybridized to mRNAs with peaks at 4 h for cytoskeletal actins and 8-12 h for smooth muscle actins. Sequential 24-h injections of the hormone produced multiple peaks of mRNA accumulation with a timing consistent with the kinetics found after a single injection of hormone. The multiple injections, however, did not result in enhanced mRNA accumulation for any of these clones. In fact, several messages showed suppressed accumulation with continued estradiol administration. Accumulated inhibitory factors in uterine cells may be responsible for this refractory condition. Except for the actin mRNA, the estradiol-stimulated mRNAs were expressed mainly in uterus and ovary. These clones may be useful in studies on the mechanism of action of estrogenic hormones and their tissue-specific regulation of gene expression.

Animals↗
PubMed1987

Immunoselection of cDNAs to avian intestinal calcium binding protein 28K and a novel calmodulin-like protein: assessment of mRNA regulation by the vitamin D hormone.

Calcium's role in a variety of cellular processes has been well documented. The storage, distribution, and delivery of calcium are regulated by a family of binding proteins including troponin C, calmodulin, parvalbumin, and vitamin D dependent calcium binding protein (CaBP-28), all of which have evolved from a common ancestral gene. To evaluate vitamin D regulation of gene transcription, a CaBP-28 cDNA (767 base pairs) was isolated from a chicken intestine lambda gt11 library utilizing a polyvalent CaBP-28 antibody as a probe. Coincident with the identification of the CaBP-28 cDNA, a group of cDNAs also was isolated (with the anti-CaBP-28 antibody) that demonstrated 84% nucleotide homology and 99% deduced amino acid homology with chicken brain calmodulin (CaM). This new CaM-like cDNA was named neoCaM. There is little nucleotide homology between the CaBP-28 cDNA and neoCaM. The CaBP-28 cDNA hybridizes with three transcripts of 2000, 2900, and 3300 bases which are dramatically induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], while the neoCaM cDNA recognizes three distinct (from CaBP-28) transcripts. Two of these mRNAs are 1400 and 1800 bases as described for brain CaM, but another larger 4000-base transcript is detected with neoCaM. Neither the CaM nor the neoCaM transcript reveals any modulation by 1,25(OH)2D3. Herein, we discuss the possible significance of not only the isolation of both cDNAs with a single antibody but also the relation of neoCaM to other well-characterized CaM cDNAs.

Amino Acid Sequence↗
PubMed1987

Hormonal regulation of rat uterine secretory protein synthesis.

Treatment of the rat uterus with estradiol results in many morphological and biochemical changes. In order to examine the biochemical mechanisms of these changes, we are interested in finding a protein which can be used as an end-point indicator or marker. Estradiol administration results in the increased synthesis and release of a 115,000 and 65,000 dalton protein into the incubation media. In this paper, we demonstrate that these proteins are actually subunits of a larger protein having a molecular weight of 180,000 daltons. This protein appears to be specific to the uterus and may be produced mainly or solely by the epithelial cells. In the normal estrous cycle, maximal production is seen at estrus with essentially no production at diestrus. In the immature rat, synthesis of the 180K dalton protein is blocked by the interaction of progesterone and estradiol. However, unlike some uterine responses, the 180K protein is increased by treatment with several antiestrogens. This protein is also present in sufficient quantities to allow for the production of monoclonal antibodies. Thus this protein has many attributes of an excellent "marker protein" and will be of value in future studies aimed at elucidating the molecular mechanism(s) of steroid hormone action in the uterus.

Animals↗
PubMed1987

Estrogen regulation of protein synthesis in the immature rat uterus: the effects of progesterone on proteins released into the medium during in vitro incubations.

We have previously identified two major medium proteins secreted from the rat uterus during in vitro incubations that appear to be estrogen regulated. In this study, immature rats were treated with estradiol (E2) progestins, and actinomycin D. Medium proteins were analyzed after incubation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. E2 (1 microgram) increased the synthesis of proteins with mol wt of 115,000 and 65,000. Progesterone inhibited this increase when given in doses of 500 and 250 micrograms and when given within 8 h of estradiol. Lower doses of progesterone were not completely inhibitory. When actinomycin D was given within 6 h of E2, it also inhibited the E2 stimulated increase. This system may provide a useful marker for monitoring hormonal action in the luminal epithelium and may help in understanding hormonal regulation of gene expression.

Animals↗
PubMed1987

Estrogen regulation of alpha 1(I)-procollagen messenger ribonucleic acid in the rat uterus.

A cDNA library, prepared from mRNA isolated from the uteri of 3-day estradiol-stimulated immature rats, was constructed in pBR322. From this library an estrogen-regulated clone, pERU3, was isolated. This clone contained sequences complementary to uterine mRNA that migrated during gel electrophoresis as a double band of about 5.0 and 5.8 kilobases. Little of this mRNA was seen in several other tissues examined. An increase in the amount of this RNA in uterus was seen 2 h after estradiol treatment, with maximum hybridization occurring, in different experiments, between 18 and 36 h, followed by a decline. Hybridization of the cDNA insert of the pERU3 plasmid with known probes indicated that it coded for alpha 1(I)-procollagen. This conclusion was supported by in vitro translation experiments in which the hybrid-selected mRNA complementary to pERU3 DNA was shown to code for a collagenase-sensitive protein with a size corresponding to that of alpha 1(I)-procollagen. This system, therefore, provides an additional tool for the study of the estrogen regulation of gene expression in the uterus.

Animals↗
PubMed1986

Estrogen regulation of protein synthesis in the immature rat uterus: the analysis of proteins released into the medium during in vitro incubation.

Immature rats were treated with estradiol (E2) or other steroids before their uteri were removed and incubated under in vitro conditions in the presence of [35S]methionine. The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the radiolabeled proteins synthesized and released into the incubation medium demonstrate that E2 regulates the appearance of two proteins. These two proteins have mol wt of 115,000 and 65,000. The concentration of proteins in the medium increases linearly with time, suggesting that they may be secreted. These two proteins were not produced by several other tissues in response to E2 and appear to be specific to the uterus. They also appear to be increased only by estrogens (E2 greater than estrone greater than estriol) and not by other steroids tested. They are increased in response to a single injection within 6 h, and the maximal concentration of proteins occurs approximately 24 h after E2 administration. The protein concentrations have essentially returned to control values by 72 h after hormone injection. The kinetics of the induction is the same for both proteins, suggesting that their increase may be coordinated. Based on the tissue and hormone specificity of the increase in the 115,000- and the 65,000-dalton proteins, they may serve as reliable markers for the study of the uterine response to E2.

Animals↗
PubMed1985

Comparison of media proteins from ovariectomized rat uteri following estrogen treatment.

Ovariectomized rats were treated with estradiol for 3 days after which their uteri were incubated in vitro and radioactive media proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Media were also chromatographed on G-25 Sephadex Blue Sepharose columns to isolate subsets of proteins. The results demonstrate that two proteins are consistently increased following estrogen treatment. These proteins have molecular weights of 104,000 and 65,000. Neither protein binds to Blue Sepharose to a great extent. The use of the protein synthesis inhibitors, emetine and actinomycin D, demonstrates that the proteins are synthesized de novo. These two proteins may serve as markers for genomic response to estradiol in the rat uterus.

Animals↗