Mandibular ossifying fibroma in a dog.

An aged mongrel dog was admitted for hemimandibulectomy as treatment for a mandibular mass that had been diagnosed as osteosarcoma. The fibro-osseous mass that surrounded the first molar tooth and replaced alveolar and cortical bone was reclassified as ossifying fibroma on the basis of anatomic location and histologic features. The tumor was composed of isomorphic fusiform cells with few mitotic figures. Tumoral stroma contained trabeculae of woven bone that were bordered by a single layer of osteoblasts. Excision was deemed complete with no evidence of extension or metastasis by computed tomography of the skull or thoracic and abdominal radiography. The dog was reportedly healthy 6 months after initial presentation. Though far less common than osteosarcoma as a primary canine bone tumor, ossifying fibroma should be included in the differential diagnosis for fibro-osseous proliferations, especially those of the jaw. Although benign, en bloc excision may be necessary for surgical cure.

Surgical treatment of simple syndactylism with secondary deep digital flexor tendon contracture in a Basset Hound.

A five-month-old, female Basset Hound was presented for lameness associated with a fused 3rd and 4th digital pad on the left hind limb (simple incomplete syndactyly), and secondary contracture of the deep digital flexure tendon of the 3rd and 4th digit. An onychectomy of the third phalanx of the third and fourth digits was performed. Following the operation, the dog gained good use of the affected limb for one month until intermittent non-weight bearing lameness developed. A second surgery was performed six months later, partially removing the second phalanx of digits three and four. Follow-up reports indicate that the dog is doing well and is without lameness. This is the first report of deep digital flexor tendon contracture and surgical treatment of this complication in canine simple syndactylism.

Syndactyly in a litter of cats.

In this case report, we describe the clinical and radiographic features of a litter of kittens affected with complex syndactyly. We also provide guidelines for the diagnosis, possible treatment and prevention of propagation of this condition. This is the first report of syndactyly in a litter of kittens and syndactyly affecting both the pectoral and pelvic limbs.

A clinical evaluation of hydroxyapatite cement in the treatment of Class III furcation defects.

BACKGROUND: Calcium hydroxyapatite cement (HAC) has been demonstrated in both animal models and human craniofacial defects to be safe, absorbable, osteoconductive, and possibly osteoinductive. This pilot study evaluated a novel technique using HAC to surgically obturate Class III mandibular molar furcation defects. METHODS: Following flap reflection, affected teeth in 6 patients were root planed and etched with citric acid. Experimental sites were grafted with HAC and coronally positioned flaps (CPF), while controls were treated by CPF only. A variety of clinical parameters were recorded initially, and at re-entry surgery 9 months later. RESULTS: At re-entry, all experimental sites exhibited granulation tissue interposed between the HAC and the alveolar bone, and clinical findings were unsatisfactory. Mean probing depth, clinical attachment loss, and recession increased by 0.8 mm, 1.9 mm, and 1.2 mm, respectively, in experimental sites. In controls, mean probing depth decreased by 0.8 mm, and clinical attachment loss and recession increased by 0.3 mm and 1.2 mm, respectively. There was a mean 1.6 mm loss in osseous crest height and a mean 2.2 mm worsening in osseous defect depth for experimental sites, but only a 0.5 mm loss in osseous crest and 0.5 mm increase in osseous defect depth in control sites. CONCLUSIONS: Experimental sites lost 1.0 to 1.5 mm in bone and attachment compared to controls, without any significant clinical benefit. While the concept of surgically obturating Class III furcation defects with a safe, osteoconductive material remains attractive, HAC did not promote repair or regeneration in this technique.

Polyunsaturated fatty acids stimulate hepatic UCP-2 expression via a PPARalpha-mediated pathway.

The discovery of homologs of the brown fat uncoupling protein(s) (UCP) UCP-2 and UCP-3 revived the hypothesis of uncoupling protein involvement in the regulation of energy metabolism. Thus we hypothesized that UCP-2 would be regulated in the hepatocyte by fatty acids, which are known to control other energy-related metabolic processes. Treatment with 250 microM palmitic acid was without effect on UCP-2 expression, whereas 250 microM oleic acid exhibited a modest eightfold increase. Eicosapentaenoic acid (EPA), a polyunsaturated fatty acid, exerted a 50-fold upregulation of UCP-2 that was concentration dependent. This effect was seen within 12 h and was maximal by 36 h. Aspirin blocked the induction of UCP-2 by EPA, indicating involvement of the prostaglandin pathway. Hepatocytes treated with arachidonic acid, the immediate precursor to the prostaglandins, also exhibited an aspirin-inhibitable increase in UCP-2 levels, further supporting the involvement of prostaglandins in regulating hepatic UCP-2. The peroxisome proliferator-activated receptor-alpha (PPARalpha) agonist Wy-14643 stimulated UCP-2 mRNA levels as effectively as EPA. These data indicate that UCP-2 is upregulated by polyunsaturated fatty acids, potentially through a prostaglandin/PPARalpha-mediated pathway.

Glucose regulation of the acetyl-CoA carboxylase promoter PI in rat hepatocytes.

The rat acetyl-CoA carboxylase (ACC) alpha gene is transcribed from two promoters, denoted PI and PII, that direct regulated expression in a tissue-specific manner. Induction of ACC, the rate-controlling enzyme of fatty acid biosynthesis, occurs in the liver in response to feeding of a high carbohydrate, low fat diet, conditions that favor enhanced lipogenesis. This induction is mainly due to increases in PI promoter activity. We have used primary cultured hepatocytes from the rat to investigate glucose regulation of ACC expression. Glucose and insulin synergistically activated expression of ACC mRNAs transcribed from the PI promoter with little or no effect on PII mRNAs. Glucose treatment stimulated PI promoter activity in transfection assays and a glucose-regulated element was identified (-126/-102), homologous to those previously described in other responsive genes, including l-type pyruvate kinase, S(14) and fatty acid synthase. Mutation of this element eliminated the response to glucose. This region of the ACC PI promoter was able to bind a liver nuclear factor designated ChoRF that interacts with other conserved glucose-regulated elements. This ACC PI element is also capable of conferring a strong response to glucose when linked to a heterologous promoter. We conclude that induction of ACC gene expression under lipogenic conditions in hepatocytes is mediated in part by the activation of a glucose-regulated transcription factor, ChoRF, which stimulates transcription from the PI promoter. Similar mechanisms operate on related genes permitting the coordinate induction of the lipogenic pathway.

Involvement of a unique carbohydrate-responsive factor in the glucose regulation of rat liver fatty-acid synthase gene transcription.

Refeeding carbohydrate to fasted rats induces the transcription of genes encoding enzymes of fatty acid biosynthesis, e.g. fatty-acid synthase (FAS). Part of this transcriptional induction is mediated by insulin. An insulin response element has been described for the fatty-acid synthase gene region of -600 to +65, but the 2-3-fold increase in fatty-acid synthase promoter activity attributable to this region is small compared with the 20-30-fold induction in fatty-acid synthase gene transcription observed in fasted rats refed carbohydrate. We have previously reported that the fatty-acid synthase gene region between -7382 and -6970 was essential for achieving high in vivo rates of gene transcription. The studies of the current report demonstrate that the region of -7382 to -6970 of the fatty-acid synthase gene contains a carbohydrate response element (CHO-RE(FAS)) with a palindrome sequence (CATGTGn(5)GGCGTG) that is nearly identical to the CHO-RE of the l-type pyruvate kinase and S(14) genes. The glucose responsiveness imparted by CHO-RE(FAS) was independent of insulin. Moreover, CHO-RE(FAS) conferred glucose responsiveness to a heterologous promoter (i.e. l-type pyruvate kinase). Electrophoretic mobility shift assays demonstrated that CHO-RE(FAS) readily bound a unique hepatic ChoRF and that CHO-RE(FAS) competed with the CHO-RE of the l-type pyruvate kinase and S(14) genes for ChoRF binding. In vivo footprinting revealed that fasting reduced and refeeding increased ChoRF binding to CHO-RE(FAS). Thus, carbohydrate responsiveness of rat liver fatty-acid synthase appears to require both insulin and glucose signaling pathways. More importantly, a unique hepatic ChoRF has now been shown to recognize glucose responsive sequences that are common to three different genes: fatty-acid synthase, l-type pyruvate kinase, and S(14).

Glucose and insulin function through two distinct transcription factors to stimulate expression of lipogenic enzyme genes in liver.

Transcription of a number of genes involved in lipogenesis is stimulated by dietary carbohydrate in the mammalian liver. Both insulin and increased glucose metabolism have been proposed to be initiating signals for this process, but the pathways by which these effectors act to alter transcription have not been resolved. We have previously defined by electrophoretic mobility shift assay a factor in nuclear extracts from rat liver, designated the carbohydrate-responsive factor (Cho- RF), that binds to liver-type pyruvate kinase and S(14) promoters at sites critical for regulation by carbohydrate. The sterol regulatory element binding protein-1c (SREBP-1c) has also emerged as a major transcription factor involved in this nutritional response. In this study, we examined the relationship between SREBP-1c and ChoRF in lipogenic gene induction. The two factors were found to possess distinct DNA binding specificities both in vitro and in hepatocytes. Reporter constructs containing binding sites for ChoRF were responsive to glucose but not directly to insulin. On the other hand, reporter constructs with an SREBP-1c site responded directly to insulin. The S(14) gene possesses binding sites for both ChoRF and SREBP, and both sites were found to be functionally important for the response of this promoter to glucose and insulin in hepatocytes. Consequently, we propose that SREBP-1c and ChoRF are independent transcription factors that mediate signals generated by insulin and glucose, respectively. For many lipogenic enzyme genes, these two factors may provide an integrated signaling system to support the overall nutritional response to dietary carbohydrate.

Inhibition of growth hormone action in models of inflammation.

Growth hormone (GH) action is attenuated during the hepatic acute-phase response (APR). To understand this attenuation, we asked whether GH and cytokine-signaling pathways intersect during an APR. In hypophysectomized rats treated with lipopolysaccharide (LPS), accumulation of activated signal transducer and transcription activator 5 (Stat5) in hepatic nuclei in response to GH and its binding to a GH response element (GHRE) from the serine protease inhibitor (Spi) 2.1 promoter are diminished in a time-dependent manner. Similarly, accumulation of activated Stat3 in hepatic nuclei in response to LPS and its binding to a high-affinity sis-inducible element (SIE) are also diminished by the simultaneous administration of GH. In functional assays with primary hepatocytes, LPS-stimulated monocyte-conditioned medium (MoCM) inhibits the GH response of Stat5-dependent Spi 2.1 reporter activity but induces Stat3-dependent Spi 2.2 reporter activity, as in an APR. Similar results are obtained when hepatocytes are treated with either tumor necrosis factor-alpha (TNF-alpha) or interleukin (IL)-1beta. TNF-alpha, IL-1beta, and IL-6 also inhibit GH-induced Spi 2.1 mRNA expression in hepatocytes. Thus inhibition of the GH signaling pathway during an APR results in reduced expression of GH-responsive genes.

An indirect role for upstream stimulatory factor in glucose-mediated induction of pyruvate kinase and S14 gene expression.

Transcription of the L-type pyruvate kinase (L-PK) and S14 genes is induced in hepatocytes in response to increased glucose metabolism. The regulatory sequences of these genes responsible for induction by glucose have been mapped to related E-box containing motifs in the promoters. Similarly, L-PK promoter activity is stimulated in a differentiated pancreatic beta-cell line, INS-1, in response to elevated glucose. By mutational analysis, we demonstrate that the sequence requirements for glucose induction in the INS-1 cell are identical to those observed in the hepatocyte, suggesting that the same transcriptional factor(s) is responsible for regulation of L-PK expression in the two cell types. One nuclear factor that binds to the glucose regulatory sequences of both of these genes is the Upstream Stimulatory Factor (USF), a ubiquitous E-box binding protein. Mice deleted for the USF2 gene display a severely delayed response to carbohydrate feeding (Vallet et al. [26]). This observation, however, does not differentiate between a direct and an indirect role for USF in the process. To gain further insight into the possible involvement of USF in glucose signaling, we have used a recombinant adenoviral construct that expresses a dominant negative form of USF. This dominant negative can dimerize with endogenous USF and is shown to inhibit DNA binding of USF in hepatocytes and INS-1 cells. However, expression of the dominant negative USF did not block the ability of glucose to stimulate L-PK or S14 gene expression in hepatocytes or L-PK promoter activity in INS-1 cells. We conclude that USF does not act by binding to the glucose regulatory sequences of the S14 or L-PK genes and the role of USF in the process of glucose induction is indirect.

Yin-yang 1 and glucocorticoid receptor participate in the Stat5-mediated growth hormone response of the serine protease inhibitor 2.1 gene.

A growth hormone-inducible nuclear factor complex (GHINF), affinity-purified using the growth hormone response element (GHRE) from the promoter of rat serine protease inhibitor 2.1, was found to contain Stat5a and -5b, as well as additional components. The ubiquitous transcription factor yin-yang 1 (YY1) is present in GHINF. An antibody to YY1 inhibited the formation of the GHINF.GHRE complex in an electrophoretic mobility shift assay. Furthermore, Stat5 was co-immunoprecipitated from rat hepatic nuclear extracts with antibodies to YY1. An examination of the GHRE shows that, in addition to two gamma-activated sites, it contains a putative YY1 binding site between the two gamma-activated sites, overlapping them both. Mutation of this putative YY1 site results in a decrease of GHINF.GHRE complex formation in an electrophoretic mobility shift assay and a corresponding decrease in growth hormone (GH) response in functional assays. The glucocorticoid receptor was also present in GHINF, and Stat5 co-immunoprecipitates with glucocorticoid receptor in hepatic nuclear extracts from rats treated with GH. GH activation of serine protease inhibitor 2.1 requires the unique sequence of the GHRE encompassing the recognition sites of several transcription factors, and the interaction of these factors enhances the assembly of the transcription complex.

Different sterol regulatory element-binding protein-1 isoforms utilize distinct co-regulatory factors to activate the promoter for fatty acid synthase.

Sterol regulatory element-binding proteins (SREBPs) activate genes of cholesterol and fatty acid metabolism. In each case, a ubiquitous co-regulatory factor that binds to a neighboring recognition site is also required for efficient promoter activation. It is likely that gene- and pathway-specific regulation by the separate SREBP isoforms is dependent on subtle differences in how the individual proteins function with specific co-regulators to activate gene expression. In the studies reported here we extend these observations significantly by demonstrating that SREBPs are involved in both sterol regulation and carbohydrate activation of the FAS promoter. We also demonstrate that the previously implicated Sp1 site is largely dispensable for sterol regulation in established cultured cells, whereas a CCAAT-binding factor/nuclear factor Y is critically important. In contrast, carbohydrate activation of the FAS promoter in primary hepatocytes is dependent upon SREBP and both the Sp1 and CCAAT-binding factor/nuclear factor Y sites. Because 1c is the predominant SREBP isoform expressed in hepatocytes and 1a is more abundant in sterol depleted established cell lines, this suggests that the different SREBP isoforms utilize distinct co-regulatory factors to activate target gene expression.

Glucose regulation of mouse S(14) gene expression in hepatocytes. Involvement of a novel transcription factor complex.

Transcription of genes encoding enzymes required for lipogenesis is induced in hepatocytes in response to elevated glucose metabolism. We have previously mapped the carbohydrate-response elements (ChoREs) of the rat liver-type pyruvate kinase (L-PK) and S(14) genes and found them to share significant sequence similarity. However, progress in unraveling this signaling pathway has been hampered due to the difficulty in identifying the key factor(s) that bind to these ChoREs. To gain further insight into the nature of the carbohydrate-responsive transcription factor, the glucose regulatory sequences from the mouse S(14) gene were examined in primary hepatocytes. Three elements were found to be essential for supporting the glucose response: a thyroid hormone-response element between -1522 and -1494, an accessory factor site between -1421 and -1392, and the ChoRE between -1450 and -1425. Of these, only the accessory factor site was conserved between the rat and mouse S(14) genes. Investigation of the ChoRE sequence indicated that two half E box motifs are critical for the response to glucose. Electrophoretic mobility shift assays revealed a complex formed between the mouse S(14) ChoRE and liver nuclear proteins. This complex was also formed by ChoREs from the rat S(14) and L-PK genes but not by mutants of these sites that are inactive in supporting the glucose response. These results suggest the presence of a novel transcription factor complex that mediates the glucose-regulated transcription of S(14) and L-PK genes.

Definition of a high affinity growth hormone DNA response element.

Rat liver contains a growth hormone inducible nuclear factor complex, GHINF, that binds to the growth hormone response element (GHRE) of the serine protease inhibitor (Spi) 2.1 gene. GHINF contains Stat5 and binds to paired gamma-activated sites (GAS) within the GHRE, but poorly to either one alone. By analysis of the sequence of various GAS sites that bind the GHINF complex (based on the GHRE 3' GAS motif), we demonstrate that a 13 nucleotide high affinity DNA recognition sequence (haGHRE) for GHINF complex binding is (ANTTC)C/T(N)A/G(GAA)A/T(A)/T. One copy of the haGHRE will replace the requirement for two GAS elements present in the wild type promoter in supporting a GH response in primary hepatocyte culture. Mutation of the native Spi 2.1 from a paired GAS site to a single haGHRE does not appreciably change its affinity for binding to the GHINF complex, nor does it alter its sensitivity to GH concentration.

Upstream stimulatory factor regulates Pdx-1 gene expression in differentiated pancreatic beta-cells.

The homeobox gene Pdx-1 plays a key role in the development of the pancreas. In the adult, however, expression of the Pdx-1 gene is restricted to pancreatic beta-cells and endocrine cells of duodenal epithelium. Recently, the transcription factor, upstream stimulatory factor (USF), has been shown to bind in vitro to a mutationally sensitive E-box motif within the 5'-flanking region of the Pdx-1 gene [Sharma, Leonard, Lee, Chapman, Leiter and Montminy (1996) J. Biol. Chem. 271, 2294-2299]. In the present study, we show that USF not only binds to the Pdx-1 gene promoter but also functionally regulates the expression of the Pdx-1 gene in differentiated pancreatic beta-cells. Adenovirus-mediated overexpression of a dominant negative form of USF2 decreased binding of endogenous USF to the E-box element by approximately 90%. This reduction in endogenous USF binding led to a greater than 50% decrease in Pdx-1 gene promoter activity, which, in turn, resulted in marked reductions in Pdx-1 mRNA and protein levels. Importantly, the lower Pdx-1 protein levels led to a greater than 50% reduction in Pdx-1 binding activity to the A3 element on the insulin gene promoter, and a significant reduction in insulin mRNA levels. Overall, our results show that USF functionally regulates Pdx-1 gene expression in differentiated pancreatic beta-cells and provide the first functional data for a role of USF in the regulation of a normal cellular gene.

Regulation of Spi 2.1 and 2.2 gene expression after turpentine inflammation: discordant responses to IL-6.

The rat serine protease inhibitor (Spi) 2 gene family includes both positive (Spi 2.2) and negative (Spi 2.1) acute phase reactants, facilitating modeling of regulation of hepatic acute phase response (APR). To examine the role of signal transducer and activation of transcription (STAT) proteins in the divergent regulation of these model genes after induction of APR, we evaluated the proximal promoters of the genes, focusing on STAT binding sites contained in these promoter elements. Induction of APR by turpentine injection includes activation of a STAT3 complex that can bind to a gamma-activated sequence (GAS) in the Spi 2.2 gene promoter, although the Spi 2.2 GAS site can bind STAT1 or STAT5 as well. To create an in vitro model of APR, primary hepatocytes were treated with combinations of cytokines and hormones to mimic the hormonal milieu of the whole animal after APR induction. Incubation of primary rat hepatocytes with interleukin (IL)-6, a critical APR cytokine, leads to activation of STAT3 and a 28-fold induction of a chloramphenicol acetyltransferase reporter construct containing the -319 to +85 region of the Spi 2.2 promoter. This suggests the turpentine-induced increase of Spi 2.2 is mediated primarily by IL-6. In contrast, although turpentine treatment reduces Spi 2.1 mRNA in vivo and IL-6 does not increase Spi 2.1 mRNA in primary rat hepatocytes, treatment of hepatocytes with IL-6 results in a 5. 4-fold induction of Spi 2.1 promoter activity mediated through the paired GAS elements in this promoter. Differential regulation of Spi 2.1 and 2.2 genes is due in part to differences in the promoters of these genes at the GAS sites. IL-6 alone fails to reproduce the pattern of rat Spi 2 gene expression that results from turpentine-induced inflammation.

Oxidant effects on epithelial Na,K-ATPase gene expression and promoter function.

The lung epithelium resorbs alveolar fluid through combined action of sodium channels and the sodium pump, Na,K-ATPase. The lung often is exposed to hyperoxia in disease states and hyperoxia generates a mixture of reactive oxygen species. In vivo and in vitro exposure of rat lung and alveolar type II cells, respectively, increases gene expression of both alpha-1 and beta-1 subunits of the sodium pump. In contrast to the primary type II cells, several type II cell lines did not increase sodium pump gene expression with hyperoxia, but the renal tubular epithelial MDCK cell line did. Using promoter-receptor constructs transfected into MDCK cells, hyperoxia did not markedly increase transcription of the alpha-1 subunit but doubled transcription of the beta-1 subunit gene. Using 5'-deletion constructs, the region required for the beta-1 increase was localized to a 40-base pair region from -44/-84. The hyperoxic responsiveness of this region was confirmed using constructs with one or two copies of this region placed in minimal promoter-luciferase reporters. This 5' promoter region contains a consensus binding sequence for SP-1, a basal transcription factor but not for binding of other known transcription factors. Thus, hyperoxia induces Na,K-ATPase beta-1 promoter transcription, likely acting through a novel mechanism.

Regulation of Na-K-ATPase gene expression by hyperoxia in MDCK cells.

Na-K-ATPase plays a central role in a variety of physiological processes, including ion transport and regulation of cell volume. Our previous data showed that hyperoxia increased the expression of Na-K-ATPase alpha 1 and beta 1 mRNA in lung type II cells. We similarly show that hyperoxia (> or = 95% O2 for 24-48 h) increased steady-state mRNA levels in both Na-K-ATPase subunits in Madin-Darby canine kidney (MDCK) cells. The mechanism of gene regulation by hyperoxia was assessed. Stability of the Na-K-ATPase mRNA levels of both subunits was unchanged in hyperoxia-exposed MDCK cells. To determine whether gene transcription was augmented by hyperoxia, MDCK cells were transfected with a beta 1-subunit promoter-reporter construct. Transfection with the wild-type promoter (beta 1-817) revealed a 1.9 +/- 0.2-fold increase in promoter activity. Transfection with 5' deletion constructs identified a 61-base pair (bp) region between -102 and -41 that was necessary for this increase in promoter activity by hyperoxia. Incorporation of this 61-bp region into a minimal promoter (mouse mammary tumor virus) similarly increased promoter activity 2.3-fold in the presence of hyperoxia. This increase in promoter activity was not seen when MDCK cells were incubated with various concentrations of hydrogen peroxide. In summary, hyperoxia increased Na-K-ATPase beta 1-subunit mRNA steady-state level due to increased transcription in MDCK cells. A region necessary for this hyperoxic effect on beta 1 transcription is located between base pairs -102 and -41 on the promoter.