Molecular cloning of canine membrane-anchored inhibitor of matrix metalloproteinase, RECK

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene is one of the endogenous matrix metalloproteinase (MMP) inhibitors. It was reported that decreased RECK expression closely correlated with tumor malignancy. We determined the cDNA sequence of the canine RECK gene. The cDNA sequence and deduced amino acid of canine RECK were 2,913 bases and 971 residues, respectively. The predicted amino acid sequence of the protein showed 95.5% and 91.9% homology with human and mouse RECK, respectively. RECK mRNA expression was analyzed in various canine tissues and tumor cell lines by quantitative RT-PCR. The highest RECK expression was detected in lung and testis. In comparison with the tissues, a remarkably low expression level was detected in tumor cell lines. In addition, the RECK gene was transfected in the canine transitional cell carcinoma, and its influence on cell proliferation, migration, and invasion was analyzed. The transfected RECK gene suppressed only canine tumor invasion. These results showed that RECK might play an important role in tumor malignancy in dogs as well as in other mammalians.     

Variations on brain microglial gene expression of MMPs, RECK, and TIMPs in inflammatory and non-inflammatory diseases in dogs

Matrix metalloproteinases (MMPs), MMP inhibitors (TIMPs, tissue inhibitors of matrix metalloproteinases), and the membrane-anchored glycoprotein RECK (reversion-inducing cysteine-rich protein with Kazal motifs) contribute to the pathogenesis of many CNS diseases. To assess the potential pathogenetic roles of microglial MMP, TIMP, and RECK generation in extracellular matrix breakdown, opening of the blood brain barrier (BBB) and subsequent recruitment of leukocytes in the CNS, twenty-four dogs suffering from spontaneously occurring different intracranial and extracranial (control group) diseases were examined. Microglia cells were isolated ex vivo by density gradient centrifugation and their expressions of MMP-2, MMP-9, MMP-12, MMP-13, MMP-14, TIMP-1, TIMP-2, and RECK were examined via quantitative real-time polymerase chain reaction (qPCR). Zymography on CNS tissues in selected cases was performed to assess differences at the protein level. Dogs were grouped in different disease categories according to histopathological examinations, in groups with or without inflammatory reactions, and in groups with/without contrast enhancement in advanced diagnostic imaging as a function of BBB breakdown. The results showed a significant up-regulation of MMP-9 in dogs with inflammation in the nervous system compared to dogs with non-inflammatory diseases. An increased expression of MMP-9 might lead to a facilitated invasion of white blood cells. Furthermore, down-regulation of MMP-13 was found in dogs with contrast enhancement. Zymographical data reflected MMP-2 qPCR data. In conclusion, differential expression of MMPs and their inhibitors, but not of RECK, which might crucially influence the pathogenesis of a given disease, could be demonstrated in canine microglia. This reflects a further pathway in the microglial repertoire to respond to various disease conditions in the CNS, a characteristic that might be of particular relevance as a target for specific treatments.     

The role of inflammation and matrix metalloproteinases in equine endometriosis

Equine endometriosis is a multifactorial disease considered to be a major cause of equine infertility. The purpose of this study was to evaluate the reliability of histomorphological grading for biopsy-like samples compared to entire uterine wall samples, to examine the association between the degree of endometriosis with animal age, and to investigate the role of inflammation in endometriosis and the expression of different matrix metalloproteinases in equine endometrium. Histomorphological lesions in 35 uterine samples were examined while comparing biopsy-like samples and entire-wall samples. Seventeen uterine samples were stained with antibodies against MMP-2, MMP-9, MMP-14, and TIMP-2. The morphologic evaluation results of the biopsy-like tissue and entire-wall samples were significantly correlated. Endometriosis in older mares (>12 years of age) was more severe than in young mares (2~4 years of age), confirming the positive correlation between animal age and disease severity, while inflammation was poorly related to the degree of endometriosis. MMP-2 and MMP-14 were detected in stromal cells, while MMP-9 and TIMP-2 were both found in stromal and glandular epithelial cells. There were no significant differences in MMPs expression between the two groups (young vs. old mares). Additional studies on the activity of MMPs could further define the role of these enzymes in equine endometriosis.     

Short-term changes of mRNA expression of various inflammatory factors and milk proteins in mammary tissue during LPS-induced mastitis

During mammary gland infection, non-specific responses are the predominant ones. The goal of this study was to investigate the mRNA expression of various soluble immune components and of the major milk proteins during the acute phase of mammary inflammation. Five healthy lactating cows were intramammary infused in one quarter with 100 microg Escherichia coli-endotoxin (lipopolysaccharide, LPS) and the contralateral quarter with saline (9 g/l) serving as control. Mammary biopsy samples of both quarters were taken immediately before and at 3, 6, 9 and 12 h after infusion and mRNA expression of various factors was quantified via real-time RT-PCR. Blood samples for determination of leukocyte number were taken simultaneously with the biopsy samples and rectal temperature was measured at 1-h intervals. Rectal temperature increased until 5h (P < 0.05) after LPS administration and remained elevated until 9 h after LPS inoculation. Blood leukocyte number decreased (P < 0.05) from 0 to 3 h from 7.7 +/- 1.1 x 10(9)l(-1) to 5.7 +/- 1.0 x 10(9)l(-1) and thereafter recovered to pre-treatment levels until 12 h after LPS challenge. In LPS-treated quarters, tumor necrosis factor-alpha and cyclooxygenase-2-mRNA expression increased (P < 0.05) to highest values at 3h after LPS challenge. Lactoferrin, lysozyme, inducible nitric oxide synthase increased (P < 0.05) and peaked at 6 h after challenge, and platelet-activating factor acetylhydrolase-mRNA expression tended to increase (P = 0.07). mRNA expression of insulin-like growth factor-I and of alphaS1-casein (CN), alphaS2-CN, beta-CN and beta-lactoglobulin did not change significantly, whereas mRNA expression of 5-lipoxygenase and alpha-lactalbumin decreased (P < 0.05) in both quarters and that of kappa-CN only in the LPS quarter. mRNA expression of some investigated factors (tumor necrosis factor-alpha, lysozyme, 5-lipoxygenase, alpha-lactalbumin) changed in control quarters, however in all respective factors less than in the LPS quarters (P < 0.05). In conclusion, mRNA expression of most inflammatory factors increased within hours, whereas that of most milk proteins remained unchanged.     

Seizure activity in dogs is associated with enhanced TIMP-2 expression of microglia

In the pathogenesis of epilepsy aberrant synaptic plasticity plays an important role. Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are responsible for nervous tissue remodelling resulting in synaptic plasticity in the central nervous system (CNS) and might therefore be crucially involved in epileptogenesis. To assess the potential pathogenetic role of microglial MMPs and TIMPs in seizure induction, twenty-four dogs suffering from different intracranial diseases with and without seizure activity were comparatively examined. Microglial cells were isolated by density gradient centrifugation and their expression profiles of MMP-2, MMP-9, MMP-12, MMP-13, MMP-14, TIMP-1, TIMP-2, and RECK (reversion-inducing cysteine-rich protein with Kazal motifs) were examined via quantitative real-time PCR (qPCR). Interestingly, a significant up-regulation of TIMP-2 expression was found for the first time in dogs suffering from seizures. In conclusion, microglial TIMP expression might be involved in seizure generation.     

Expression of matrix metalloproteinases in bovine luteal cells induced by prostaglandin F2α, interferon γ and tumor necrosis factor α

We recently demonstrated that luteal cells flow out from the ovary via lymphatic vessels during luteolysis. However, the regulatory mechanisms of the outflow of luteal cells are not known. Matrix metalloproteinases (MMPs) can degrade the extracellular matrix and basal membrane, and tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit the activity of MMPs. To test the hypothesis that MMP expression in luteal cells is regulated by luteolytic factors, we investigated the effects of prostaglandin F2α (PGF), interferon γ (IFNG) and tumor necrosis factor α (TNF) on the mRNA expression of MMPs and TIMPs in cultured luteal cells. Luteal cells obtained from the CL at the mid-luteal stage (days 8–12 after ovulation) were cultured with PGF (0.01, 0.1, 1 μM), IFNG (0.05, 0.5, 5 nM) and TNF (0.05, 0.5, 0.5 nM) alone or in combination for 24 h. PGF and IFNG significantly increased the expression of MMP-1 mRNA. In addition, 1 μM PGF in combination with 5 nM IFNG stimulated MMP-1 and MMP-9 mRNA expression significantly more than either treatment alone. In contrast, IFNG significantly decreased the level of MMP-14 mRNA. The mRNA expression of TIMP-1, which preferentially inhibits MMP-1, was suppressed by 5 nM INFG. One μM PGF and 5 nM IFNG suppressed TIMP-2 mRNA expression. These results suggest a new role of MMPs: luteal MMPs stimulated by PGF and IFNG break down the extracellular matrix surrounding luteal cells, which accelerates detachment from the CL during luteolysis, providing an essential prerequisite for outflow of luteal cells from the CL to lymphatic vessels.     

Activity of matrix metalloproteinase-2 (MMP-2) in canine oronasal tumors

Activity of matrix metalloprotease-2 (MMP-2) and the expression of its related molecules were examined in spontaneous canine oronasal tumors. Tissue samples from melanoma and squamous cell carcinoma possessed higher MMP-2 activity, as shown in gelatin zymography, in comparison with acanthomatous epulis and nasal adenocarcinoma. Regional lymph node invasion and distant metastases were more frequently observed in the MMP-2 positive cases. There were no significant differences by RT-PCR examination in the expression of the genes encoding MMP-2, MT1-MMP and TIMP-2 among the tumor histological types. However, the MMP-2/TIMP-2 ratio showed a significantly higher level of the genes in the malignant oral melanoma and squamous cell carcinoma. The MMP-2/TIMP-2 ratio was also positively correlated with MMP-2 activity in gelatin zymography. These results indicate that the MMP-2/TIMP-2 ratio may be of value in evaluating the prognosis in canine oronasal cavity tumors.     

Influence of persistent canine distemper virus infection on expression of RECK, matrix-metalloproteinases and their inhibitors in a canine macrophage/monocytic tumour cell line (DH82)

A morbillivirus infection of tumour cells is known to exert oncolytic activity, but the mechanism of this inhibitory action has not been well defined. Matrix metalloproteinases (MMPs) are important enzymes degrading the extracellular matrix and are often upregulated in malignant neoplasms. Recent studies have demonstrated that RECK may potently suppress MMP-2 and -9 activity, thus inhibiting angiogenesis and metastasis. In this study, real time quantitative polymerase chain reaction (RT-qPCR) was used to determine the effect of persistent infection with canine distemper virus (CDV) infection on the expression of MMPs and their inhibitors (TIMPS) in a canine macrophage/monocytic tumour cell line (DH82). The activity of proMMP-2 and proMMP-9 was also verified zymographically. Following CDV infection, MMP-2, TIMP-1 and TIMP-2 were down-regulated, while RECK was upregulated. These findings suggest that CDV infection restores RECK expression in tumour cells and may interfere with the intracellular processing of MMPs and TIMPs, thus possibly influencing tumour cell behaviour beneficially for the host. However, this needs to be verified in in vivo studies.     

MMP-9 as a marker of inflammation in tracheal epithelial lining fluid (TELF) and in bronchoalveolar fluid (BALF) of COPD horses

Gelatinolytic activity was analysed to study whether elevated activity previously found at the tracheal level of the respiratory tract of horses with chronic obstructive pulmonary disease (COPD) could also be found at the lower part of the respiratory tract. Furthermore, presence and significance of the gelatinolytic matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in respiratory secretions of healthy and COPD horses were determined. Elevated gelatinolytic matrix metalloproteinases were detected in bronchoalveolar and tracheobronchial secretions from COPD horses. The main pathologically elevated MMP was characterised to be MMP-9. Significantly increased MMP-9 activities as measured by gelatin zymography and Western blotting were found in all the respiratory samples from COPD horses compared to healthy horses. Elevation of active MMP-9 paralleled with increased gelatinase-associated lipocalin levels. Bronchoalveolar lavage fluid (BALF) epithelial cells, macrophages, neutrophils and lymphocytes expressed MMP-9 immunoreactivity demonstrated by immunocytochemistry and MMP-9 mRNA was expressed by bronchial epithelial cells of lung tissue section shown by in situ hybridisation. MMP-2 seems not to play a major role in chronic lung inflammation. No clear differences in MMP-2 or MMP-14 (a potent MMP-2 activator) levels were found when comparing the samples from COPD or healthy horses. These results suggests that MMP-9 could serve as a potential diagnostic marker for the active ongoing tissue remodelling in the acute phase of equine COPD. Increased gelatinolytic activity could be found at both tested respiratory tract levels. Therefore, tracheal epithelial lining fluid (TELF) samples can usefully serve as diagnostic material for detection of increased levels of the main gelatinolytic MMP, MMP-9, representing the entire diseased lung.     

Matrix metalloproteinase mRNA expression in canine anal furunculosis lesions

Although the aetiology of anal furunculosis (AF) in dogs is poorly understood, there is evidence for an underlying immune dysfunction. This is illustrated by the presence of a T helper type 1 cytokine mRNA profile in AF lesions and the clinical response to ciclosporin therapy. Expression of MMPs 2, 9 and 13 were evaluated in AF lesional biopsies by real-time quantitative RT-PCR. There was significantly increased expression of both MMP-9 and MMP-13 mRNA in AF biopsies compared to controls (p<0.001) but no significant difference in MMP-2 mRNA expression. Since MMP-9 and MMP-13 are primarily produced by macrophages, these data suggest that ulceration could be the result of aberrant activation of this cell type in the tissues. It is feasible that such pathological macrophage activity occurs in response to interferon-gamma secreted by T helper type 1 cells. This could explain why the lesions resolve following treatment with the immunosuppressive drug ciclosporin.     

九龙牦牛PPARγ基因的克隆及其表达谱分析

根据普通牛(Bos tarus)过氧化物酶体增殖物激活受体γ(PPARγ)基因序列设计引物,用成年九龙牦牛(Bos grunniens)脂肪组织总RNA,经RT-PCR扩增获得了PPARγ基因序列(GenBank登陆号:GU061328),其中cDNA的ORF为1 428 bp,编码475个氨基酸,与普通牛PPARγ氨基酸的同源性达99%,有2个氨基酸发生突变。利用半定量RT-PCR分析九龙牦牛PPARγ基因的mRNA表达特性。结果表明:在脂肪、背最长肌、心、肝、肾、脾和肺脏中均检测到PPARγ基因的表达,并且在脂肪组织中表达量极显著高于其他组织(P0.01),在肝脏和脾脏中亦有较高表达。PPARγ基因在背最长肌中的表达5.5岁九龙牦牛显著高于0.5、3.5岁和9岁以上,其表达与背最长肌的肌内脂肪含量未见显著相关性。     

子宫内膜基质金属蛋白酶-2、-9 mRNA表达与奶牛子宫内膜炎关系的研究

为探讨基质金属蛋白酶(MMPs)-2、MMP-9在奶牛子宫内膜炎中的作用机制,选用产后6～10d健康及患急性化脓性子宫内膜炎的中国荷斯坦奶牛各10头,分别为对照组和试验组,通过ELISA检测PGF2a、孕酮浓度;免疫组化检测子宫内膜Ⅰ型胶原、Ⅳ型胶原的表达;SYBR Green I荧光定量PCR检测子宫内膜MMP-2和MMP-9mRNA表达。结果表明:试验组PGF2a浓度极显著低于对照组(P<0.01),孕酮含量显著高于对照组(P<0.05),PGF2a与孕酮浓度呈显著负相关(r=0.893);试验组子宫内膜中Ⅰ型胶原、Ⅳ型胶原的表达量均高于对照组,差异极显著(P<0.01);MMP-2和MMP-9mRNA水平则显著低于对照组(P<0.01)。结果提示:产后子宫内膜炎病牛子宫内膜MMP-2、MMP-9低表达,导致Ⅰ型和Ⅳ型胶原降解受阻,使细胞外基质为子宫内膜细胞提供的刺激信号不足,子宫分泌PGF2a量少,黄体消退障碍,血清孕酮含量高;说明子宫内膜MMP-2、MMP-9mRNA表达对产后子宫内膜炎的发生和发展起促进作用。     

Relationship between retinoic acid receptor alpha gene expression and growth-inhibitory effect of all-trans retinoic acid on canine tumor cells

Retinoids show antitumor effects on human acute promyelocytic leukemia and other tumors via retinoid receptors. In dogs, the role of retinoid receptors in inhibiting tumor development remains unclear. To evaluate the correlation between the degree of expression of retinoic acid receptor alpha (RARalpha) mRNA and the antiproliferative effects of all-trans retinoic acid (ATRA) treatments, expression analysis of RARalpha mRNA and cell growth inhibition assay were performed on 17 established canine tumor cell lines, including 6 mammary gland tumor (MGT) cell lines, 3 osteosarcoma cell lines, 5 melanoma cell lines, and 3 mast cell tumor (MCT) cell lines. Among the cell lines investigated, all 3 MCT cell lines showed high expression of RARalpha, and the most effective cell growth inhibition was observed in ATRA-treated MCT cell lines. However, remarkable antiproliferative effects of ATRA treatments were not observed on other tumor cell lines with moderate or low RARalpha mRNA expression. As a result of the relationship between RARalpha mRNA expression and ATRA treatment with regression analysis, statistically significant correlation was suggested. Furthermore, real-time quantitative polymerase chain reaction analysis of RARalpha was performed on MCT tissue samples of dogs with spontaneous disease, and 5 of 9 tissues showed high expression. These results suggest that ATRA may be an effective antitumor agent for MCT in dogs, and that prior measurement of expression of RARalpha mRNA may be a good indicator of the effectiveness of ATRA treatment.     

Expression patterns of the BRCA1 splicing variants in canine normal tissues and mammary gland tumors

Human BRCA1 is familial breast cancer susceptibility gene. Recently, decreased BRCA1 mRNA and protein expression has been identified in sporadic breast tumors. In the reported human BRCA1 splicing variants, delta11b lacks the majority of exon11 and is suspected to have a distinct function in normal tissues. The splicing variants display a variety of expression pattern in breast cancer samples. Although mammary gland tumor is important disease in dog, there are few reports for BRCA1 in the canine tumors. In this study, we examined the relative amounts of BRCA1 splicing variants mRNA in canine normal and mammary tumor samples by RT-PCR to investigate whether there is the altered expression of variant mRNAs in the canine tumor as reported in human. The exon11b-defecting RT-PCR products were observed in all the normal tissues examined and the nucleotide sequence was quite similar to that of human BRCA1 delta11b. In some tumor samples, we did not detect the products targeted for exon10-13 and exon14-15, while these products were observed in all the normal samples examined. Especially, the relative amounts of the exon11-defecting products were remarkably decreased in most of the tumors (11/16).