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B R Troen

Publications and source records attributed to B R Troen.

At least 19 recordsLinked to original sources
PubMed2001

New insights into the regulation of cathepsin K gene expression by osteoprotegerin ligand.

Cathepsin K plays a key role in bone resorption. We provide the first evidence that osteoprotegerin ligand (OPGL), a critical pro-resorptive cytokine, acutely stimulates the expression of cathepsin K in osteoclasts. We used in situ RT-PCR and real time quantitative RT-PCR to analyze cathepsin K gene expression. OPGL enhanced cathepsin K mRNA levels in mature osteoclasts isolated from rat neonatal long bones. OPGL together with macrophage colony-stimulating factor (M-CSF) also stimulated cathepsin K gene expression in monocytic cells and multinucleate osteoclasts in bone marrow cultures. Real time quantitative RT-PCR demonstrated high levels of cathepsin K mRNA in bone marrow cultures, paralleling the degree of osteoclastogenesis. We therefore suggest that OPGL enhances bone resorption, at least in part, by inducing cathepsin K gene expression.

Animals↗
PubMed2001

Macrophage colony-stimulating factor suppresses osteoblast formation.

We provide the first evidence that the bone marrow-derived cytokine, macrophage colony-stimulating factor (M-CSF), inhibits the formation of bone-forming osteoblasts. We examined both osteoclast and osteoblast formation in primary rat bone marrow cultures. As expected, M-CSF together with osteoprotegerin ligand (OPGL) markedly accelerated osteoclastogenesis. In contrast, treatment with M-CSF alone yielded no osteoclasts at any time. The most striking and novel observation was that M-CSF with or without OPGL dramatically suppressed osteoblast formation. In separate experiments, estradiol markedly suppressed osteoclast formation in the M-CSF/OPGL-treated cultures independently of osteoblasts. Consistent with this was the expression of estrogen receptor-alpha (ERalpha) and ERbeta mRNA in osteoclast precursors. We therefore conclude that in addition to the well-known action of M-CSF to modulate osteoclastogenesis, this cytokine may also regulate osteoblast formation.

Animals↗
PubMed2000

Transcription of human cathepsin B is mediated by Sp1 and Ets family factors in glioma.

Cathepsin B expression is increased at both the mRNA and protein levels in a wide variety of tumors. The mechanisms responsible for this regulation are not well elucidated. We have isolated a 2.2-kb cathepsin B genomic fragment that contains the 5'-flanking region of the cathepsin B gene. Using reporter gene analysis in human glioblastoma U87MG cells, we have mapped a 228-bp fragment (-172 to +56) having high promoter activity. This promoter region has a high G+C content; contains potential Spl, Ets, and USF binding motifs; and lacks canonical TATA and CAAT boxes immediately upstream of the major transcriptional initiation site. Cotransfection experiments demonstrated that Spl and Ets1 could trans-activate cathepsin B transcription, whereas Ets2 could not. Electrophoretic mobility shift assays and supershift assays revealed that three of the four putative Sp1 sites in this promoter region form a specific complex containing the Sp1 transcription factor. Mutating all four of the Spl binding sites individually markedly reduced the promoter activity of transfected reporter genes in U87 cells. Cotransfection of this cathepsin B promoter construct with Spl family expression vectors in Schneider's Drosophila line 2 (SL2) cells demonstrated that Spl and Sp3, but not Sp4, activated cathepsin B transcription. Taken together, these results suggest that Sp1, Sp3, and Ets1 are important factors in cathepsin B transcription. The regulation of cathepsin B transcription by Sp1- and Sp1-related factors is mediated through multiple GC boxes.

Base Sequence↗
PubMed1999

CD38/ADP-ribosyl cyclase: A new role in the regulation of osteoclastic bone resorption.

The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.

ADP-ribosyl Cyclase↗
PubMed1999

Differentiating agents regulate cathepsin B gene expression in HL-60 cells.

We utilized HL-60 cells as a model system to examine the regulation of ctsb gene expression by differentiating agents. Inducers of monocytic differentiation [phorbol ester (PMA), calcitriol (D3), and sodium butyrate (NaB)] and inducers of granulocytic differentiation [all-trans retinoic acid (RA) and 9-cis retinoic acid (9-cis RA)] increase ctsb mRNA levels in a dose-dependent manner as determined by Northern blot hybridization. D3 and retinoids exert additive effects, suggesting that these agents act in part through distinct pathways. Actinomycin D decay experiments indicate that D3, NaB, RA, and 9-cis RA do not alter mRNA stability. In contrast, PMA markedly increases the half-life of ctsb mRNA. In transient transfection assays, PMA and NaB both stimulate transcription of the luciferase reporter gene placed under the control of ctsb promoter fragments. Thus, inducers of HL-60 cell differentiation can regulate the expression of the ctsb gene at both transcriptional and posttranscriptional levels.

Alitretinoin↗
PubMed1995

Comparative responsiveness of HL-60, HL-60R, and HL-60R+ (LRARSN) cells to retinoic acid, calcitriol, 9 cis-retinoic acid, and sodium butyrate.

In HL-60 cells, retinoic acid (RA) and 9 cis-RA induce granulocytic differentiation, and calcitriol and sodium butyrate induce monocytic differentiation. To study the role of retinoid resistance on the response to these agents, we investigated their effects in HL-60 cells, retinoid-resistant HL-60R cells, and HL-60R+ cells in which retinoid sensitivity has been restored. In HL-60 cells, cathepsin D (ctsd) mRNA levels are increased by these agents and by cholera toxin after pretreatment with each agent. Calcitriol, 9 cis-RA, and sodium butyrate increase interleukin-8 (IL-8) mRNA expression, and pretreatment with these agents or RA potentiates the stimulation of IL-8 by phorbol ester (TPA). Pretreatment of HL-60 cells with all of the agents confers inducibility of cathepsin L (ctsl) mRNA by TPA in previously unresponsive cells. In HL-60R cells, none of the agents alone or in combination significantly enhances the expression of the ctsd, IL-8, or ctsl mRNAs. Retinoid stimulation (either alone or in combination with the other agents) of the three mRNAs is partially restored in the HL-60R+ cells. Calcitriol does not alter the expression of any of these mRNAs, and only the stimulation of IL-8 mRNA by sodium butyrate is recovered. Treatment with all of the agents inhibits proliferation and stimulates differentiation of the HL-60 cells. RA and calcitriol are unable to inhibit proliferation of the HL-60R cells, whereas only calcitriol fails to inhibit proliferation of the HL-60R+ cells. None of the agents induces differentiation in either the HL-60R or HL-60R+ cells. Therefore, the mutation of the RA receptor alpha is insufficient to account for the altered responses of the HL-60R cells, and there are likely defects in other signaling pathways in these cells. These cells may prove useful in examining the mechanism of cross-resistance between various differentiating agents.

Butyrates↗
PubMed1995

Regulation of cathepsin D gene expression in HL-60 cells by retinoic acid and calcitriol.

Cathepsin D (ctsd) is a lysosomal acid protease found in neutrophils and monocytes. We investigated whether differentiating agents increase the expression of ctsd mRNA in HL-60 cells. Treatment with either retinoic acid or calcitriol enhances the steady-state levels of ctsd mRNA in a dose-dependent manner. The stimulation by retinoic acid requires new protein synthesis. Pretreatment with retinoic acid enhances the response of the ctsd gene to prostaglandin E2. To determine whether the effects of retinoic acid and calcitriol are associated with differentiation, we pretreated Hl-60 cells for 120 h with inducers of granulocytic differentiation (lithium chloride, DMSO, and retinoic acid) and monocytic differentiation (calcitriol, sodium butyrate, and phorbol ester). Lithium chloride and DMSO do not significantly affect ctsd mRNA expression, and none of the granulocytic inducers alters the subsequent response of the ctsd gene to calcitriol. All of the monocytic inducers stimulate ctsd mRNA, and both calcitriol and sodium butyrate significantly potentiate the subsequent response to retinoic acid. Transcription initiation of the ctsd gene occurs at one major and several minor sites and is unaffected by treatment with retinoic acid and calcitriol or pretreatment with other differentiating agents. Although differentiation appears to influence ctsd mRNA expression, calcitriol and retinoic acid stimulate ctsd gene expression via mechanisms that are independent of their role in differentiation.

Base Sequence↗
PubMed1995

Phorbol ester stimulated cathepsin L expression in U937 cells.

Cathepsin L (ctsl) is a lysosomal cysteine proteinase, the synthesis and secretion of which is induced by transformation, growth factors, and tumor promoters. We studied the effect and the mechanism of action of phorbol ester (TPA) on the expression of ctsl mRNA in U937 histiocytic leukemia cells. TPA treatment induces ctsl mRNA in a manner that is dose-dependent, occurs at the level of transcription, and is ablated by cotreatment with cycloheximide but is unaffected by dexamethasone. Treatment with TPA plus staurosporine, a potent protein kinase C inhibitor, results in greater expression of ctsl mRNA than does treatment with TPA alone. Similar to TPA, staurosporine alone increases ctsl transcription, an effect that is inhibited by cycloheximide. Another PKC inhibitor, H7, exerted no effect upon the induction of ctsl mRNA by either TPA or staurosporine. Staurosporine and H7, however, inhibit the increase in c-jun mRNA by TPA. In contrast, the tyrosine kinase inhibitors herbimycin A and genistein inhibit the effect of TPA and staurosporine upon ctsl mRNA with little or no effect on c-jun expression. Pretreatment with sodium orthovanadate enhances the induction of ctsl expression by TPA and staurosporine. These data suggest that, in U937 cells, TPA-stimulated ctsl gene transcription is apparently activated by a protein kinase C-independent signal transduction pathway involving tyrosine kinase activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

Cloning, genomic organization, and chromosomal localization of human cathepsin L.

Cathepsin L is a lysosomal cysteine protease whose expression and secretion is induced by malignant transformation, growth factors, and tumor promoters. Many human tumors express high levels of cathepsin L, which is a broad spectrum protease with potent elastase and collagenase activities. Two published human cathepsin L cDNA sequences differ only in their 5'-untranslated regions. In this study, we demonstrate the concurrent expression of two distinct human cathepsin L mRNAs (hCATL-A and hCATL-B) in adenocarcinoma, hepatoma, and renal cancer cell lines. Cloning of the human cathepsin L gene by polymerase chain reaction amplification of genomic DNA and subsequent sequencing reveals that hCATL-A and hCATL-B mRNAs are encoded by a single gene. The 3' end of the first intron contains the 5' portion of hCATL-B and is contiguous to the second exon of the gene. These data suggest either the possibility of alternative splicing or the presence of a second promoter within the first intron of the hCATL gene. We mapped the hCATL gene to chromosome 9q21-22. Sequencing of both the mouse and human cathepsin L genes demonstrates almost complete conservation of exon and intron position, but significant divergence in intron structure, possibly reflecting differences in regulation of expression of the mouse and human cathepsin L genes.

Amino Acid Sequence↗
PubMed1991

Downstream sequences mediate induction of the mouse cathepsin L promoter by phorbol esters.

The major excreted protein (MEP) of mouse fibroblasts is the precursor to a lysosomal acid protease (cathepsin L) whose synthesis is induced by malignant transformation, growth factors, tumor promoters, and cyclic AMP. We have previously cloned a functional gene for MEP from NIH 3T3 cells. When subcloned into chloramphenicol acetyl transferase (CAT) expression vectors, both 4-kilobase and 300 base pair fragments in the 5'-flanking region of the MEP gene confer CAT activity that is stimulated by cyclic AMP treatment but is not stimulated by phorbol ester treatment of NIH 3T3 cells. These fragments confer constitutive promoter activity that is comparable to that of the SV40 promoter. Primer extension, using RNA from cells transiently transfected with MEP-CAT fusion plasmids, demonstrates that phorbol ester treatment increases the amount of transcript from constructs containing both the promoter and sequences downstream of the transcription initiation site, including the first three introns, but not from constructs containing only the 5'-flanking region of the MEP gene. Nuclear run-off experiments confirm that the increase in endogenous MEP mRNA is mediated by increased transcription and not via relief of transcriptional attenuation. Since both the MEP promoter, which contains three potential binding sites for the AP-2 transcription factor, and the SV40 promoter, which contains both AP-1 and AP-2 binding sites, fail to respond to 12-O-tetradecanoylphorbol-13-acetate in NIH 3T3 cells, these upstream motifs are not sufficient to confer phorbol ester responsiveness in NIH 3T3 cells. These results suggest that the MEP gene is regulated in a complex manner by sequences both upstream and downstream of the transcription initiation site.

Animals↗
PubMed1988

Cloning and expression of the gene for the major excreted protein of transformed mouse fibroblasts. A secreted lysosomal protease regulated by transformation.

The major excreted protein (MEP) of mouse fibroblast cells is the 39,000 Mr precursor to a lysosomal acid protease (cathepsin L) induced by malignant transformation, growth factors, and tumor promoters. We have cloned and characterized the gene for MEP from NIH-3T3 cells. This cosmid clone (pcosMMEP), containing the unique 12,000-base pair mouse MEP gene, has been transfected into monkey kidney (CV-1) cells and human epidermoid carcinoma (A431) cells. The stable A4MEP transfectants produce mouse MEP that is an active cathepsin which is secreted, glycosylated, and processed intracellularly to lower molecular weight forms as in the wild-type NIH-3T3 cells. The CVMEP cells (nontransformed phenotype) produce quantities of mouse MEP similar to that found in NIH-3T3 cells, whereas the A4MEP cells (transformed phenotype) produce greater amounts of MEP similar to the levels seen in Kirsten virus-transformed NIH-3T3 cells. The MEP mRNAs from both mouse cells and stably transfected human cells are the same size and have the same single major site for initiation of transcription, indicating that the cloned mouse MEP promoter is active in transfected cells.

Animals↗
PubMed1988

Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease.

Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

Animals↗
PubMed1987

Sequence and expression of the cDNA for MEP (major excreted protein), a transformation-regulated secreted cathepsin.

The major excreted protein (MEP) of malignantly transformed mouse fibroblasts is a secreted thiol proteinase. Sequencing of the MEP cDNA shows the coding region for the protein to be identical with the sequence for a mouse cysteine proteinase isolated from macrophages, but the MEP cDNA is polyadenylated at a different site in the 3' non-coding region. Strong homology of MEP with human cathepsin L suggests that MEP is the mouse analogue of cathepsin L. Amino acid sequencing of the N-terminus of the secreted form of MEP indicates that, during secretion, the polypeptide is cleaved between amino acids 17 and 18. We have placed the MEP cDNA in a eukaryotic expression vector and demonstrated the production of the 39 kDa polypeptide form of mouse MEP in monkey CV-1 cells.

Amino Acid Sequence↗
PubMed1986

Effect of age on the 3,5,3'-tri-iodothyronine-induced increase in sugar uptake in rat thymocytes.

The effect of age on the responsiveness of rat thymocytes to 3,5,3'-tri-iodothyronine (T3) was studied. It has been demonstrated previously that the plasma membrane-mediated effect of T3 to increase sugar uptake by rat thymocytes is influenced by age and sex. In both sexes, T3 given in vitro stimulated sugar uptake in cells from animals of 15 days of age, had no effect at 21 days and was again effective at 26 days. In the male, thymocytes from animals of 40 days of age and older were refractory to T3. However, in the female, T3, although less effective than in cells from 26-day-old animals, remained stimulatory in cells from 40- and 60-day-old rats. T3 had no effect in cells from animals of 90 days of age and older. In in-vivo studies in which female rats of 26, 60 and 90 days of age were first injected with T3 and 1 h later with [3H]2-deoxyglucose, the responsiveness of thymocytes to T3 also declined progressively with advancing age; T3 was most effective in cells from 26-day-old animals, less stimulatory in 60-day-old and essentially without effect in cells from 90-day-old animals. From these observations we have concluded that in both male and female rats the responsiveness of thymocytes to T3 declines progressively with age, and that this decline occurs at an earlier age in cells obtained from males.

Aging↗
PubMed1985

Effects of age and sex on certain metabolic functions and mitogenic activity in rat thymocytes.

In female and male Sprague-Dawley CD rats, both total mass and thymocyte content of thymus gland peaked during the second month of life and then declined with age. Values of both 2-deoxyglucose (2-DG) uptake and thymidine incorporation in thymocytes were higher in cells from females than from males in all age groups studied, these differences being significant only at 3 months of age and onward. In cells from both sexes, thymocyte 2-DG uptake and thymidine incorporation were at maximum at the first and second months of age, respectively. Thereafter, in cells from males, 2-DG uptake and thymidine incorporation declined rapidly with advancing age; whereas in cells from females, these functions started to decline only after 6 months. In both freshly isolated and cultured cells, the concanavalin A (Con A)-induced increase in thymidine incorporation was also affected by age, peaking at about 3 months. The proportionate effect of Con A on thymidine incorporation was similar in cells of both sexes, but because of differences in basal values, the absolute increase in thymidine incorporation produced by Con A was higher in cells from females than from males. Since thymocytes are the progenitors of T-cells, these changes in the number and function of thymocytes may be the forerunner of similar functional abnormalities that are seen in circulating T-cells, but at a later age.

Age Factors↗
PubMed1982

Influence of age and sex on the concentrations of thyroid hormone in serum in the rat.

Studies of the influence of age and sex on the concentrations of total thyroxine (T4) and 3,5,3'-tri-iodothyronine (T3) in serum and on the free T4 and free T3 indices, were conducted in Sprague-Dawley rats of the CD strain varying in age between 10 days and 12 months. Both sex- and age-related differences were found. In all age-groups studied, serum T4 concentrations were higher in the male than in the female, whereas serum T3 concentrations were higher in the female. In both sexes, concentrations of T4 and T3 in serum reached a peak early in life, between the first and second month of age, and declined thereafter. In addition, in both sexes the intensity of thyroid hormone binding, as judged from values of the in-vitro uptake of T3, did not change with age, suggesting that free T4 and T3 concentrations in the serum display the same sex differences and age-related changes as do the concentrations of total T4 and T3. It remains to be determined whether these sex- and age-related alterations in serum thyroid hormone concentration are expressed in differences in the activity of various thyroid hormone-dependent processes.

Aging↗