Correction to: Lower grip strength and dynamic body balance in women with distal radial fractures.

The article Lower grip strength and dynamic body balance in women with distal radial fractures, written by. K. Fujita, H. Kaburagi, A. Nimura, T. Miyamoto, Y. Wakabayashi, Y. Seki, H. Aoyama, H. Shimura, R. Kato, A. Okawa was originally published electronically on the publisher's internet portal.

Lower grip strength and dynamic body balance in women with distal radial fractures.

In this case-control study, we concluded that women with distal radial fractures who were surgically treated showed lower grip strength and dynamic body balancing than those of controls. These results suggest that measurements of grip strength and dynamic body balance may be useful screening tools to assess future fracture risk. INTRODUCTION: Patients with distal radial fractures (DRFs) are at risk of future fragility fractures. However, their physical characteristics and tendencies for falls remain unclear. We aimed to compare the physical characteristics of women with and without distal radial fractures. METHODS: We included 128 women with a DRF as their first fragility fracture (fracture group) who underwent surgical treatment. Concurrently, 128 age- and sex-matched participants without a history of fragility fractures were selected as controls (control group). The participants underwent assessments of grip strength and the body balancing ability test. Measurements were taken twice in the fracture group, at 2 weeks and 6 months postoperatively, and once in the control group. The body balancing ability test included the Functional Reach Test, Timed Up and Go test (TUG), 2-Step test (2ST), and Timed Uni-pedal Stance test. The participants also completed questionnaires about their health. RESULTS: There were no significant differences (p > 0.05) in patient characteristics between the groups. The fracture group showed lower grip strength across all age groups. In the DRF group, prolonged TUG time was observed at 2 weeks postoperatively in all age groups and at 6 months in participants aged 55-74 years; the 2ST score was significantly lower in participants aged between 65 and 74 years. CONCLUSIONS: Women with DRF demonstrated lower grip strength and dynamic body balancing ability. Lower grip strength and dynamic body balancing ability were identified as significant risk factors in women with DRF, suggesting that these may be useful screening tools to assess fracture risk.

Research and development productivity map: visualization of industry status.

WHAT IS KNOWN AND OBJECTIVE: Decline in research and development (R&D) productivity and changes in the business environment have led to pharmaceutical company management to strive to improve R&D productivity. This decline is widely considered to be a major cause of industry consolidation and has received increased scholarly attention. This study aims to construct an R&D productivity map to visualize the industry's R&D productivity and to identify similarity in corporate actions with a view to investigate whether there is a relationship between deterioration in R&D productivity and industry consolidation. METHODS: Research and development productivity is decomposed into two subprocesses to measure productivity: R&D efficiency and R&D effectiveness, and scores were calculated using a two-stage data envelopment analysis (DEA). The map is then constructed by projecting outputs. To identify any relationship between DEA scores and merger and acquisition transactions, a multiple regression model is employed. RESULTS AND DISCUSSION: Data on 21 global pharmaceutical companies, statistical results indicated that companies with lower R&D efficiency scores were more likely to engage in consolidation. Three US companies that were least successful in terms of R&D effectiveness, as measured by our indicators, were either acquired or changed their business model. CONCLUSION: The R&D productivity map is a useful means for visualizing productivity among companies. By grouping companies into four groups, behavioural commonalities can be observed. The R&D productivity map should be useful for monitor the industry's productivity and help to improve it.

Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase.

Autosomal recessive juvenile parkinsonism (AR-JP), one of the most common familial forms of Parkinson disease, is characterized by selective dopaminergic neural cell death and the absence of the Lewy body, a cytoplasmic inclusion body consisting of aggregates of abnormally accumulated proteins. We previously cloned PARK2, mutations of which cause AR-JP (ref. 2), but the function of the gene product, parkin, remains unknown. We report here that parkin is involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating enzyme UbcH7, and that mutant parkins from AR-JP patients show loss of the ubiquitin-protein ligase activity. Our findings indicate that accumulation of proteins that have yet to be identified causes a selective neural cell death without formation of Lewy bodies. Our findings should enhance the exploration of the molecular mechanisms of neurodegeneration in Parkinson disease as well as in other neurodegenerative diseases that are characterized by involvement of abnormal protein ubiquitination, including Alzheimer disease, other tauopathies, CAG triplet repeat disorders and amyotrophic lateral sclerosis.

Ubiquitination of a new form of alpha-synuclein by parkin from human brain: implications for Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive accumulation in selected neurons of protein inclusions containing alpha-synuclein and ubiquitin. Rare inherited forms of PD are caused by autosomal dominant mutations in alpha-synuclein or by autosomal recessive mutations in parkin, an E3 ubiquitin ligase. We hypothesized that these two gene products interact functionally, namely, that parkin ubiquitinates alpha-synuclein normally and that this process is altered in autosomal recessive PD. We have now identified a protein complex in normal human brain that includes parkin as the E3 ubiquitin ligase, UbcH7 as its associated E2 ubiquitin conjugating enzyme, and a new 22-kilodalton glycosylated form of alpha-synuclein (alphaSp22) as its substrate. In contrast to normal parkin, mutant parkin associated with autosomal recessive PD failed to bind alphaSp22. In an in vitro ubiquitination assay, alphaSp22 was modified by normal but not mutant parkin into polyubiquitinated, high molecular weight species. Accordingly, alphaSp22 accumulated in a non-ubiquitinated form in parkin-deficient PD brains. We conclude that alphaSp22 is a substrate for parkin's ubiquitin ligase activity in normal human brain and that loss of parkin function causes pathological alphaSp22 accumulation. These findings demonstrate a critical biochemical reaction between the two PD-linked gene products and suggest that this reaction underlies the accumulation of ubiquitinated alpha-synuclein in conventional PD.

Intractable ulcer caused by Mycobacterium shinshuense: successful identification of mycobacterium strain by 16S ribosomal RNA 3'-end sequencing.

An extremely rare case of intractable ulcer caused by Mycobacterium shinshuense is described. A 59-year-old Japanese woman developed an ulcerated subcutaneous induration on the upper arm. Ziehl-Neelsen staining revealed positive bacilli. Tissue culture isolated Mycobacterium species, but standard identification techniques (including molecular biological approaches such as DNA-DNA hybridization) could not distinguish the precise causative pathogen, although it was narrowed down to three possibilities: Mycobacterium marinum, Mycobacterium ulcerans and M. shinshuense. Finally, a novel 16S rRNA sequencing method enabled the diagnosis of M. shinshuense infection. The epidemiology of the cutaneous infection caused by this mycobacterium has yet to be elucidated, but a review of reported cases indicated that ulcers having some resemblance to those caused by M. ulcerans infection were found in nonendemic areas and that M. shinshuense could be considered as the cause. The approach introduced in this report could provide a powerful tool for the identification of this organism.

HGF/NK4, a four-kringle antagonist of hepatocyte growth factor, is an angiogenesis inhibitor that suppresses tumor growth and metastasis in mice.

We reported that NK4, composed of the N-terminal hairpin and subsequent four kringle domains of hepatocyte growth factor (HGF), acts as the competitive antagonist for HGF. We now provide the first evidence that NK4 inhibits tumor growth and metastasis as an angiogenesis inhibitor as well as an HGF antagonist. Administration of NK4 suppressed primary tumor growth and lung metastasis of Lewis lung carcinoma and Jyg-MC(A) mammary carcinoma s.c. implanted into mice, although neither HGF nor NK4 affected proliferation and survival of these tumor cells in vitro. NK4 treatment resulted in a remarkable decrease in microvessel density and an increase of apoptotic tumor cells in primary tumors, which suggests that the inhibition of primary tumor growth by NK4 may be achieved by suppression of tumor angiogenesis. In vivo, NK4 inhibited angiogenesis in chick chorioallantoic membranes and in rabbit corneal neovascularization induced by basic fibroblast growth factor (bFGF). In vitro, NK4 inhibited growth and migration of human microvascular endothelial cells induced by bFGF and vascular endothelial growth factor (VEGF) as well as by HGF. HGF and VEGF activated the Met/HGF receptor and the KDR/VEGF receptor, respectively, whereas NK4 inhibited HGF-induced Met tyrosine phosphorylation but not VEGF-induced KDR phosphorylation. NK4 inhibited HGF-induced ERK1/2 (p44/42 mitogen-activated protein kinase) activation, but allowed for bFGF- and VEGF-induced ERK1/2 activation. These results indicate that NK4 is an angiogenesis inhibitor as well as an HGF antagonist, and that the antiangiogenic action of NK4 is independent of its activity as HGF antagonist. The bifunctional properties of NK4 to act as an angiogenesis inhibitor and as an HGF antagonist raises the possibility that NK4 may prove therapeutic for cancer patients.

Selective cytotoxicity of phospholipids and diacylglycerols to rat 3Y1 fibroblasts transformed by adenovirus type 12 or its E1A gene.

The colony-forming ability of rat 3Y1 fibroblasts transformed by adenovirus type 12 (Ad12) was drastically reduced when the cells were cultivated for 18 h in medium augmented with 300 micrograms/ml of liposomes composed of either phosphatidylcholine (PC) or phosphatidylinositol. In contrast, those of untransformed 3Y1 cells and simian virus 40-transformed and polyomavirus-transformed 3Y1 cells were not. The cytotoxicity of PC liposomes was also observed in 3Y1 cells transformed by plasmid DNA containing Ad12-E1A gene but not in those transformed by adenovirus type 2, Rous avian sarcoma virus, or plasmid DNA carrying v-Ha-ras oncogene. The extensive killing of Ad12-transformed and E1A-transformed 3Y1 cells occurred in liposomes of dioleoyl-PC and of dilinoleoyl PC but not those of dipalmitoyl PC, distearoyl-PC, or diarachidonyl PC, suggesting that the acyl groups of phospholipids play an important role in cytotoxicity. Dilinoleoylglycerol, 60 micrograms/ml, was also cytotoxic selectively to Ad12-transformed and E1A-transformed 3Y1 cells, although the toxicity of lysophosphatidylcholine or linoleic acid was not specific to these transformants. These results suggest that cell transformation by Ad12 is characterized by a high sensitivity to exogenously administered phospholipids and diacylglycerol that contain oleoyl or linoleoyl acyl groups and that the sensitivity is attributable to the expression of E1A gene of Ad12.

Mechanism of selective killing by dilinoleoylglycerol of cells transformed by the E1A gene of adenovirus type 12.

Rat 3Y1 fibroblasts transformed by the E1A gene of adenovirus type 12 (E1A-3Y1 cells) are highly sensitive to the cell-killing effect of 1,3-dilinoleoylglycerol (DLG) administered in a culture medium, whereas the parental 3Y1 cells are less sensitive (H. Shimura et al., Cancer Res., 48: 578-583, 1988). The selective cytotoxicity of DLG to E1A-3Y1 cells was markedly reduced by the simultaneous administration of nonspecific antioxidants such as vitamin E, butylated hydroxytoluene, and ascorbic acid. Specific scavengers for oxygen radicals had no effect. Lipoxygenase inhibitors (nordihydroguaiaretic acid, esculetin, and baicalein) reduced the DLG-mediated selective cytotoxicity, whereas cyclooxygenase inhibitors (acetylsalicylic acid and indomethacin) showed no effect. The intracellular and extracellular contents of the products from lipid peroxidation as measured by the thiobarbituric acid test were significantly greater in E1A-3Y1 cells than in the parental 3Y1 cells. In comparison with DLG, linoleic acid and monolinoleoylglycerol were equally toxic to E1A-3Y1 and parental 3Y1, and trilinoleoylglycerol was weakly toxic to both types of cells. Scanning electron microscopy revealed that numerous holes about 0.2 micron in diameter were scattered all over the surface of the E1A-3Y1 cells after treating the cultures with DLG. These results suggest that; (a) the DLG-mediated cytotoxicity to the E1A-transformed cells is attributable to lipid peroxidation; (b) the structural property of DLG is essential to the E1A specificity of cytotoxicity; and finally (c) the destruction of the cell membrane is the basis of cytotoxicity of DLG.

Transcriptional activation of the thyroglobulin promoter directing suicide gene expression by thyroid transcription factor-1 in thyroid cancer cells.

Gene therapy with thyroglobulin (TG) promoter and a prodrug/suicide gene combination may prove useful as a treatment for thyroid carcinoma. However, most poorly differentiated and anaplastic thyroid carcinomas have lost the ability to express the TG gene expression accompanied by loss of transcription factors [thyroid transcription factor-1 (TTF-1), TTF-2, or Pax-8] interacting with the TG promoter. In anticipation of developing transcriptionally targeted gene therapy of TG-nonproducing thyroid carcinomas, we investigated the effect of TTF-1 gene transfer on TG promoter activity and the cytotoxic effect obtained by the TG promoter-driven HSV-TK gene along with ganciclovir in thyroid carcinoma and nonthyroidal cells. Using a chimeric construct containing the 5'-flanking region of the rat TG gene between -826 and +39 bp and the luciferase gene, TG promoter activity was detected in a normal rat thyroid cell line (FRTL-5), but not in a dedifferentiated line of thyroid cells (FRT) expressing Pax-8 but not TTF-1, TTF-2, or TG [TTF-1(-)/TTF-2(-)/Pax-8(+)/TG(-)], or in a human papillary thyroid carcinoma cell line [BHP15-3; TTF-1(-)/TTF-2(-)/Pax-8(-)/TG(-)], a human pulmonary cell line [H441; TTF-1(+)/TTF-2(-)/Pax-8(-)/TG(-)], or a dog kidney epithelial cell line [MDCK; TTF-1(-)/TTF-2(-)/Pax-8(+)/TG(-)]. Cotransfection of the TTF-1 expression vector stimulated TG promoter activity in FRT and BHP15-3 dedifferentiated thyroid cells, but not in H441 pulmonary cells. Only weak activation was observed in MDCK kidney cells. We then constructed recombinant adenovirus vectors, AdTTF-1 and ADTGTK: AdTTF-1 contained cytomegalovirus promoter and rat TTF-1 cDNA; AdTGTK carried the TG promoter-driven HSV-TK gene. Infection with AdTGTK and combined with GCV treatment induced a cytotoxic effect in FRTL-5 cells but not in dedifferentiated thyroid or nonthyroid cells. Cotransduction of AdTTF-1 and AdTGTK permitted 90% cytotoxicity for BHP15-3 and >95% cytotoxicity for FRT, as well as for BHP7-13 and BHP18-21v thyroid cancer cell lines [both/TTF1(-)/TTF-2(-)/Pax-8(+)/TG(-)]. In contrast, little cytotoxicity was seen for H441 and MDCK cell lines even with 300 microg/ml of ganciclovir. These results suggest that cotransduction of a TG promoter-controlled suicide gene and the TTF-1 gene by adenoviral vectors confers transcriptionally targeted gene-mediated cytotoxicity in poorly differentiated thyroid carcinoma cells unable to express the TG gene.

Inhibition of tumor growth and invasion by a four-kringle antagonist (HGF/NK4) for hepatocyte growth factor.

Invasion of various carcinoma cells follows their interaction with stromal cells. Hepatocyte growth factor (HGF), four-kringle-containing growth factor, is a mesenchymal or stromal-derived mediator which affects the growth and the invasiveness of carcinoma cells. We now have evidence that a four-kringle-containing antagonist for HGF, HGF/NK4 inhibits invasion of tumors in vivo, as well as in vitro. HGF/NK4 competitively inhibited the binding of HGF to Met/ HGF receptors on GB-d1 human gallbladder carcinoma cells. HGF induced invasion of the cells through Matrigel basement membrane components and into collagen gels, but HGF-induced invasion was inhibited by HGF/NK4. Invasion of GB-d1 cells was induced by co-cultivation with stromal fibroblasts, which mimics tumor-stromal interaction, but it was almost completely suppressed by HGF/NK4. Likewise, invasive growth induced by HGF in collagen gels in GB-dl cells, HuCC-T1 human cholangiocarcinoma cells, and ME-180 human uterus cervical carcinoma cells was also strongly inhibited by HGF/NK4. When GB-d1 cells were implanted subcutaneously into nude mouse, tumor cells invaded muscular tissue, but the infusion of HGF/NK4 inhibited this invasion. Furthermore, HGF/NK4 increased apoptotic cell death of GB-d1 cells and inhibited tumor growth in vivo. These results indicate that HGF/ NK4 may inhibit growth and invasion of carcinoma cells, as mediated by HGF during tumor-stromal interactions. We propose that there is a unique therapeutic potential for HGF/NK4 to prevent tumor invasion and perhaps even metastasis.

Telomerase activity in pancreatic juice differentiates ductal carcinoma from adenoma and pancreatitis.

Telomerase activity was measured in pancreatic juice obtained by endoscopic retrograde pancreatography from 34 patients (12 with ductal carcinoma, 12 with pancreatic adenoma, and 10 with pancreatitis). The activity in pancreatic juice was expressed as the number of cells of a human pancreatic cancer cell line, MIA PaCa-2, that exhibit an activity equal to that expressed in 1 microg of protein from pancreatic juice. A telomerase ladder was detected in the pancreatic juice obtained from a majority of the patients with ductal adenocarcinoma. The median value of relative telomerase activity in the carcinoma samples was 9.38 (25th percentile, 3.14; 75th percentile, 95.8), a value significantly higher than that derived from patients with either pancreatitis or pancreatic adenoma (P < 0.0001). When a threshold value of relative telomerase activity of 3.00 was used, 75% (9 of 12) of the samples obtained from patients with ductal carcinoma were positive. We conclude that telomerase activity in pancreatic juice differentiates adenocarcinoma from adenoma and pancreatitis and may serve as a useful diagnostic tool.

Telomerase elevation in pancreatic ductal carcinoma compared to nonmalignant pathological states.

Telomerase activity was measured in surgically resected tissues of 20 human pancreatic ductal carcinomas, 12 adenomas, 5 pancreatitis tissues, 14 normal pancreatic ducts, and 13 normal pancreatic tissues (primarily made up of acinar cells) using a PCR-based telomerase assay. Relative telomerase activity was expressed as the equivalent telomerase intensity of the number of cells of a human pancreatic cancer cell line, MIA PaCa-2, per microgram of protein in the tissue samples. The median value (25th percentile, 75th percentile) of relative telomerase activity in pancreatic carcinomas was 13.2 (3.58, 244), which was significantly higher relative to normal tissues, normal ducts, pancreatitis tissues, and adenomas (P < 0.0001). When the cutoff value of relative telomerase activity was set at 1.00 and 3.00, the positivity rates of telomerase activity in pancreatic ductal carcinomas were 100 and 80%, respectively. Some of the adenoma samples displayed a weak telomerase ladder. However, when semiquantitatively analyzed, the relative telomerase activity of all adenoma tissues was less than 1.00 equivalent cells per microgram protein of the tissues, which was equivalent to the values encountered in normal ducts. Thus, our results indicate that reactivation of telomerase may occur at a late stage of pancreatic ductal carcinogenesis. Therefore, telomerase may be a specific marker for distinguishing pancreatic cancer from pancreatitis and adenomas.

Parkin is linked to the ubiquitin pathway.

Autosomal recessive juvenile parkinsonism (AR-JP) is one of the most common forms of familial Parkinson's disease. AR-JP is characterized by selective and massive loss of dopaminergic neurons in the substantia nigra of the midbrain and absence of Lewy bodies, the pathological hallmark of idiopathic Parkinson's disease. Parkin, the causative gene of AR-JP, encodes a 52-kDa protein that is a RING-type ubiquitin (Ub) protein ligase (E3) collaborating with a Ub-conjugating enzyme (E2) belonging to a cognate class of UbcH7 or UbcH8. Analysis of parkin mutations in AP-JP patients reveals that the functional loss of parkin as an E3 enzyme is the molecular basis of AR-JP. Thus it is now clear that AR-JP is due to failure of proteolysis mediated by the Ub-proteasome system and accumulation of as yet unidentified protein(s) causes nigral neuronal death without formation of Lewy bodies. These findings should shed new light on the mechanisms underlying neurodegeneration in sporadic Parkinson's disease as well as AR-JP.

Single strand DNA-binding proteins and thyroid transcription factor-1 conjointly regulate thyrotropin receptor gene expression.

An element, -186 to -176 base pairs (bp), in the minimal TSH receptor (TSHR) promoter binds thyroid transcription factor-1 (TTF-1) and is important for both constitutive expression and TSH/cAMP-induced negative autoregulation of the TSHR in thyroid cells. An element on the noncoding strand of the TSHR, contiguous with the 5'-end of the TTF-1 element, has single strand binding activity. It is distinct from the TTF-1 site, as evidenced by competition experiments using gel shift assays; but the association of the two elements is not random. Thus, the single strand binding protein (SSBP) element also exists contiguous to the 5'-end of an upstream TTF-1 site, -881 to -866 bp; mutation of two conserved nucleotides in each SSBP element results in the loss of SSBP binding and cross-competition. Transfection experiments indicate that full, constitutive TSHR gene expression in FRTL-5 thyroid cells requires the binding of both SSBPs and TTF-1, since mutation of either element halves thyroid-specific promoter activity, whereas mutation of both decreases promoter activity to values near those of a control vector. Transfection experiments with rat liver cells support their independent activities and show that the SSBP site contributes to TSHR gene expression in non-thyroid tissue. The SSBPs function conjointly with TTF-1 in thyroid-specific, TSH/cAMP-induced negative autoregulation of the TSHR. Thus, TSH or forskolin-treated FRTL-5 cells coordinately decrease TSHR RNA levels and TSHR DNA binding to both the SSBPs and TTF-1; also the maximal TSH/cAMP-induced decrease in gene expression requires both elements. The TSH-induced effect in each case is inhibited by cycloheximide; the TSH-induced decrease in SSBP/DNA complex formation requires the presence of insulin or calf serum, exactly as does TSH-induced down-regulation of TSHR RNA levels. In sum, full, constitutive expression of the TSHR in thyroid cells requires TTF-1 and the SSBPs to bind separate, contiguous elements on the TSHR promoter. TSH/cAMP decreases the binding of each factor to its respective site, thereby decreasing TSHR gene expression. The role of the SSBP and TTF-1 sites in constitutive TSHR expression and in TSH/cAMP-induced negative regulation of the TSHR is, therefore, additive and independent.

Role of the cyclic adenosine 3',5'-monophosphate response element in efficient expression of the rat thyrotropin receptor promoter.

The "minimal" promoter region of the TSH receptor gene, -195 to -39 basepairs (bp), exhibits basal promoter activity, thyroid specificity, and negative regulation by TSH via its cAMP signal. In FRT thyroid cells and by comparison to pTRCAT5'-199, 5'-deletion mutants of chloramphenicol acetyltransferase (CAT) constructs from -199 to -150 bp of the minimal promoter decrease basal CAT activity by 50%, whereas continued deletion to -146 bp increases activity more than 4-fold. Continued deletion to -131 bp results in basal activity less than that of the -199 bp construct. An octameric cAMP response element (CRE)-like sequence, TGAGGTCA, is within -146 to -131 bp and starts at -139 bp. Its mutation to a consensus CRE (TGACGTCA) or AP1 (TGAGTCA) site or mutation of several residues flanking its 3'-terminus can improve promoter activity as much as 8-fold compared to pTRCAT5'-199. A nonpalindromic mutation to CGAGGACA decreases basal promoter activity to the level of the 199-bp minimal promoter. The CRE-like sequence between -139 and -132 bp is a constitutive enhancer of promoter activity in FRT thyroid cells, since, ligated to a simian virus-40-promoter-driven CAT gene, it increases CAT activity in the absence of forskolin in proportion to copy number and independent of direction or position. It can, however, function as a cAMP-responsive CRE, as evidenced by the fact that forskolin increases the activity of the same simian virus-40-promoter-driven CAT gene constructs in Buffalo rat liver (BRL) cells. DNAase-I footprinting shows that the CRE region is protected by a purified binding region peptide of the CRE-binding protein, activating transcription factor-2, and recombinant AP1 (human c-jun) as well as by BRL, FRT, and FRTL-5 rat thyroid cell nuclear extracts. Gel mobility shift analyses show that multiple CRE-binding proteins in the BRL, FRT, and FRTL-5 cell nuclear extracts form complexes with the CRE-like site, that one of these is CRE-binding protein, and that all form complexes with mutant sequences of the CRE-like site in a manner that exactly parallels their effects on constitutive enhancer function in FRT thyroid cells. We show, therefore, that the CRE-like site in the minimal TSH receptor promoter functions as a constitutive enhancer of promoter activity in FRT thyroid cells yet is a cAMP-responsive CRE.(ABSTRACT TRUNCATED AT 400 WORDS)

A Y-box protein is a suppressor factor that decreases thyrotropin receptor gene expression.

The decanucleotides in a tandem repeat, -162 to -140 bp, are suppressor elements that decrease TSH receptor (TSHR) gene expression by different mechanisms. A factor(s) interacting with the 3'-decanucleotide compete for proteins that bind the cAMP response element, -139 to -132 bp, a constitutive enhancer necessary for efficient TSHR expression. The 5'-decanucleotide is in a CT-rich, S1 nuclease-sensitive region of the promoter; its suppressor activity has been related to its ability to bind a nonthyroid-specific protein to its coding strand. In this report we clone a complementary DNA encoding a single strand DNA-binding protein that forms a specific protein-DNA complex with the coding strand of the 5'- but not the 3'-decanucleotide and not with the 5'-decanucleotide noncoding or double strand. We show, by cotransfection with TSHR promoter-chloramphenicol acetyltransferase chimeras, that the protein is a suppressor that regulates the function of the 5'- but not the 3'-decanucleotide. The protein is a Y-box protein that was previously cloned as an enhancer factor from the rat liver; it is, however, 95% identical to human YB-1, which suppresses major histocompatibility class II gene expression, and to human nuclease-sensitive element protein-1, a Y-box protein identified by its ability to bind single strand, CT-rich, nuclease-sensitive elements of genes that, like the TSHR, have GC-rich promoters. Unexpectedly, the Y-box protein binds two other sites in the minimal TSHR promoter in a single strand-specific fashion and acts a suppressor at each of these sites. One is associated with the insulin response element of the minimal TSHR promoter and is not in an overtly CT-rich region. The other is located 3' to the cAMP response element in a region termed the S-box, -120 to -113 bp, because of its homology to the S-box of the major histocompatibility class II promoter; this site is in a CT-rich area and, as in the class II promoter, is linked to cAMP-induced gene suppression. A conserved CCTC sequence in each site is important for the binding and suppressor function of the Y-box protein.

Analysis of differentiation-induced expression mechanisms of thyrotropin receptor gene in adipocytes.

Rat adipose tissue, as well as differentiated 3T3-L1 cells, has been shown to express TSH receptor (TSHR) mRNA in amounts approaching those in the thyroid. We investigated the molecular mechanisms of TSHR gene expression in adipose cells. Primer extension and cloned cDNA sequences showed that transcription of the TSHR gene in rat adipose tissue was from multiple start sites clustered between -89 to -68 bp and almost identical to those in FRTL-5 thyroid cells. By transient expression analysis, we localized, between -146 and -90 bp, a positive regulatory element, the activity of which was markedly increased after the differentiation of 3T3-L1 cells. Deoxyribonuclease I protection showed that nuclear extracts from differentiated 3T3-L1 cells strongly protected two sequences, from -146 to -127 bp, including a cAMP response element-like sequence and from -112 to -106 bp containing a putative Ets-binding sequence. In differentiated 3T3-L1 cells, disruption or deletion of either sequence was found to result in the loss of enhancer activity, suggesting both elements may synergistically activate the TSHR promoter. Electrophoretic mobility shift analysis revealed the induction of new protein/DNA complexes formed either with the cAMP response element-like site or with putative Ets elements after the differentiation into adipocytes. In contrast, nuclear proteins, whose binding to DNA was diminished after the differentiation of 3T3-L1 cells, were found to interact with the site contiguous to the 5'-end of the putative Ets-binding sequence. Mutations of this binding site, which reduced the protein/DNA complex formation, increased TSHR promoter activity in undifferentiated cells. These observations suggested that differentiation-induced diminution of suppressor interactions may allow the enhancers to synergistically activate the transcription of TSHR gene in adipocytes.

Regulation of the rat thyrotropin receptor gene by the methylation-sensitive transcription factor GA-binding protein.

The GA-binding protein (GABP), a transcription factor with a widespread tissue distribution, consists of two subunits, a and beta1, and acts as a potent positive regulator of various genes. The effect of GABP on transcription of the TSH receptor (TSHR) gene in rat FRTL-5 thyroid cells has now been investigated. Both deoxyribonuclease I footprint analysis and gel mobility-shift assays indicated that bacterially expressed glutathione S-transferase fusion proteins of GABP subunits bind to a region spanning nucleotides (nt) -116 to -80 of the TSHR gene. In gel mobility-shift assays, nuclear extracts of FRTL-5 cells and FRT cells yielded several specific bands with a probe comprising nt -116 to -80. Supershift assays with antibodies to GABPalpha and to GABPbeta1 showed that GABP was a component of the probe complexes formed by the nuclear extracts. Immunoblot analysis confirmed the presence of both GABP subunits in the nuclear extracts. A reporter gene construct containing the TSHR gene promoter was activated, in a dose-dependent manner, in FRTL-5 cells by cotransfection with constructs encoding both GABPalpha and GABPbeta1. Both GABP binding to and activation of the TSHR gene promoter were prevented by methylation of CpG sites at nt -93 and -85. These CpG sites were highly methylated (>82%) in FRT cells and completely demethylated in FRTL-5 cells, consistent with expression of the TSHR gene in the latter, but not the former. These results suggest that GABP regulates transcription of the TSHR gene in a methylation-dependent manner and that methylation of specific CpG sites and the methylation sensitivity of GABP contribute to the failure of FRT cells to express the endogenous TSHR gene.

Thyroid-specific expression and cyclic adenosine 3',5'-monophosphate autoregulation of the thyrotropin receptor gene involves thyroid transcription factor-1.

The chimeric chloramphenicol acetyltransferase (CAT) construct, pTRCAT5'-199, containing the TSH receptor (TSHR) minimal promoter, -199 to -39 base pairs (bp), exhibits the thyroid specificity and TSH/cAMP autoregulation evident in TSHR gene expression. The present report shows that a cis-acting element between -189 and -175 bp, which binds thyroid transcription factor-1 (TTF-1), is involved in both activities. The 22 bp between -199 and -178 contains a positive element important for expression of the TSHR minimal promoter in rat FRTL-5 thyroid cells. DNAase I footprinting shows that extracts from functioning FRTL-5, but not non-functioning FRT thyroid or Buffalo rat liver (BRL) cells, protect a region between -189 and -175 bp. The protection is duplicated by TTF-1, and the protected element has only a two-base mismatch from the consensus TTF-1 element identified in the thyroglobulin (TG) and thyroid peroxidase minimal promoters. Gel mobility shift analyses reveal that FRTL-5 thyroid cell nuclear extracts form a specific protein/DNA complex with this region, which is prevented by the TTF-1 binding element from the TG promoter; FRT and BRL cell nuclear extracts do not have TTF-1 and do not form this complex. A role for the TSHR/TTF-1 binding element in thyroid-specific expression of the TSHR gene is evidenced as follows. Overexpression of TTF-1 in FRT or BRL cells, which have no TTF-1, increased the activity of pTRCAT5'-199, but not pTRCAT5'-177, which has no TTF-1 binding element. A nonsense mutation of the TTF-1 binding element eliminated TTF-1-induced activation of TSHR promoter activity in FRT or BRL cells and reduced TSHR promoter activity in FRTL-5 thyroid cells. In contrast, mutation of this element to the TTF-1 consensus sequence of the TG or thyroid peroxidase promoter had no significant influence on TSHR promoter activity. The activity of the TSHR/TTF-1 binding element requires a functioning cAMP response element (CRE). Thus, TTF-1 activity is lost when the CRE site is mutated to a nonfunctional, nonpalindromic sequence; it is, in contrast, maximized when CRE activity is maximized by its mutation to a consensus AP1 element. TTF-1 phosphorylation is important for binding and activity. Thus, binding of TTF-1 to the TSHR/TTF-1 element is phosphatase-sensitive and is increased by treating nuclear extracts with the catalytic subunit of protein kinase A. Overexpression of the catalytic subunit of PKA enhances TTF-1-increased activity of the TSHR minimal promoter.(ABSTRACT TRUNCATED AT 400 WORDS)