Expression of fugu TIMP-3 and -4 genes in adult tissues and embryos

The tissue inhibitors of metalloproteinases (TIMPs) are involved in various processes of extra-cellular matrix (ECM) metabolism by inhibiting matrix metalloproteinases (MMPs). However, the fundamental information for these genes is little known in fish. Previously, we report cDNA cloning and gene expressions of two fugu (Takifugu rubripes) TIMP-2s. Here, we cloned cDNA of fugu TIMP-3 and performed an expression analysis of TIMP-3 and -4 mRNA in fugu adult tissues using a quantitative real-time PCR. The expression level of TIMP-3 mRNA was constitutive in all tissues, while TIMP-4 was significantly higher in the brain (P=0.05). Further, we performed a whole mount in situ hybridization in fugu embryos at different stages. In early stages, TIMP-3 mRNA was abundant in the somites and the caudal end of the notochord. At hatching larvae, the TIMP-3 mRNA was abundant in the pectoral fin, dorsal and ventral fin fold along the entire antero-posterior axis. TIMP-3 may be involved in axis elongation and somitogenesis. TIMP-4 mRNA was expressed in the tail bud, at the midbrain-hindbrain boundary and in the diencephalon from 72 to 104 hpf. This indicates TIMP-4 is highly expressed in the brain matrix in vivo.     

Expression and distribution of fugu TIMP-2s (fgTIMP-2a and fgTIMP-2b) mRNAs in tissues and embryos

In teleosts, two distinct types of TIMP-2s occur, TIMP-2a and TIMP-2b, but little is known about their locations and quantitative expressions. Here, we examined pufferfish (Takifugu rubripes) TIMP-2a (fgTIMP-2a) and TIMP-2b (fgTIMP-2b) quantities and locations in fugu adult tissues and embryos. To compare the quantitative expression of fgTIMP-2s, we performed a quantitative real-time PCR (qPCR). FgTIMP-2a mRNA was constitutively expressed and significant differences in expression were not observed among adult tissues. Whereas, fgTIMP-2b mRNA was significantly differently expressed in ordinary muscle and gill compared to the expression level in whole blood (P<0.05). Although significant difference was not observed between brain and other tissues, both fgTIMP-2s mRNAs were abundant in the brain. In addition, we examined embryos during development using qPCR. Both fgTIMP-2s mRNAs gradually increased during embryonic development from 48 hpf. However, fgTIMP-2b mRNA was obviously abundant compared to fgTIMP-2a mRNA in embryos. We also examined the specific mRNA distribution in embryos. The fgTIMP-2s mRNAs showed the same distribution during development. Both fgTIMP-2s are expressed in adult fugu tissues and embryos but their expression levels clearly differ, suggesting that there is a predominance of fgTIMP-2b over fgTIMP-2a in vivo.     

鲤鱼发育早期HPG轴和GH/IGF轴相关因子的转录起始分析

采用RT-PCR的方法,以不同发育时期的鲤鱼胚胎和幼鱼为材料,研究了与鱼类生殖相关的HPG轴以及与生长相关的GH/IGF轴中GnRH、GtH以及GH、GHR和IGF重要信号分子的转录起始特征.结果显示,sGnRH、cGnRH、GtH-Ⅰβ卢亚基和GHR于鲤鱼胚胎受精后20h开始转录,IGF-1于受精后23h开始转录,GtH-Ⅱβ亚基于受精后26h开始转录,GtH α亚基于受精后46h开始转录,GH于1dph(孵出后第1天)开始转录.其中,GHR和IGF-1均早于GH开始转录,GtH α亚基和β亚基的转录起始时间不同步.研究结果为揭示鲤鱼生殖与生长间的调控机制积累了重要的科学依据.     

MMP-2、MMP-7、MMP-9、TIMP-1及TIMP-2在乳腺癌组织中的表达及意义

目的:探讨基质金属蛋白酶及其抑制剂在乳腺癌组织中的表达及其与肿瘤浸润转移的关系,为乳腺癌的临床治疗及预后预测提供基础。方法:选择我院2012年5月至2014年5月收治的乳腺癌患者80例,对所选病例的乳腺癌组织、癌旁组织及正常乳腺组织样本进行检测。观察并比较不同乳腺组织中MMP-2,MMP-7、MMP-9、TIMP-1及TIMP-2 m RNA的表达水平。结果:与正常乳腺组织相比较,乳腺癌组织和癌旁组织中MMP-2、MMP-7、MMP-9,TIMP-1及TIMP-2 m RNA的表达显著增加,差异具有统计学意义(P0.05)。乳腺癌组织中MMP-2、MMP-7、MMP-9、TIMP-1及TIMP-2 m RNA的表达显著高于癌旁组织和正常组织,差异具有统计学意义(P0.05)。随着肿瘤范围扩大,MMP-2、MMP-7和MMP-9 m RNA的表达水平显著增加(P0.05),而TIMP-1和TIMP-2 m RNA表达无显著变化(P0.05)。随着淋巴结转移进展,MMP-2、MMP-7和MMP-9 m RNA的表达显著增加(P0.05),而TIMP-1和TIMP-2 m RNA无显著变化(P0.05)。结论:MMP-2、MMP-7、MMP-9、TIMP-1和TIMP-2的m RNA在乳腺癌组织中呈高表达,这可能与乳腺癌的发生和发展有关,而MMP-2、MMP-7和MMP-9可能有助于预测乳腺癌的侵袭行为。     

斑马鱼整体原位杂交法研究MMP15a的发育相关表达的初步观察

克隆斑马鱼基质金属蛋白酶15a（MMP15a）基因,并研究其在斑马鱼胚胎早期发育中的时空表达状况。收集不同发育时期的斑马鱼胚胎,制备DIG标记的MMP15a RNA探针,采用全胚胎原位杂交方法研究MMP15a基因在胚胎斑马鱼的表达。结果MMP15a基因在胚胎受精后一个细胞时期就开始表达,从受精后24h起,在眼睛处表达明显,从受精后48h MMP15a在胸鳍和耳囊有特异性表达至到受精后96h。MMP15a在斑马鱼胚胎发育不同时期表达明显,且在胸鳍和耳囊处有持续表达。     

Expressions of Raldh3 and Raldh4 during zebrafish early development

Retinoic acid (RA) plays crucial roles in vertebrate embryogenesis. Four retinal dehydrogenases (Raldhs) that are responsible for RA synthesis have been characterized in mammals. However, only Raldh2 ortholog is identified in zebrafish. Here, we report the identification of raldh3 and raldh4 genes in zebrafish. The predicted proteins encoded by zebrafish raldh3 and raldh4 exhibit 70.0% and 73.5% amino acid identities with mouse Raldh3 and Raldh4, respectively. RT-PCR analyses reveal that both raldh3 and raldh4 mRNAs are present in early development. However, whole mount in situ hybridization shows that raldh3 mRNA is first expressed in the developing eye region of zebrafish embryos at 10-somite stage. At 24 hpf (hours post fertilization), raldh3 mRNA is expressed in the ventral retina of eye. At 36 hpf, the mRNA is also expressed in otic vesicle in addition to ventral retina, and it maintains its expression pattern till 2 dpf (days post fertilization). At 3 dpf, raldh3 mRNA becomes very weak in ventral retina but is present in otic vesicle at a high level. At 5 dpf and 7 dpf, raldh3 is no longer expressed in eyes but is expressed in otic vesicles at a very low level. raldh4 mRNA is initially detected in developing liver and intestine regions at 2 dpf embryos. Its expression level becomes very high in these two regions of embryos from 3 dpf to 5 dpf. Analysis on the sections of 5 dpf embryos reveals that raldh4 is expressed in the epithelium of intestine. At 7 dpf, raldh4 mRNA is only weakly expressed in the epithelium of intestinal bulb.     

整体原位杂交法初步研究MMP11b在斑马鱼胚胎发育过程中的表达

克隆斑马鱼基质金属蛋白酶11b（MMP11b）基因,并研究其在斑马鱼胚胎早期发育中的时空表达状况。收集不同发育时期的斑马鱼胚胎,制备DIG标记的MMP11b RNA探针,采用全胚胎原位杂交方法研究MMP11b基因在斑马鱼胚胎的表达。MMP11b基因在胚胎受精后一个细胞时期就开始表达,并且一直持续到96h,从受精后24h起,在耳囊处表达明显,在受精后48h时期在胸鳍和肛门处也有特异性表达。MMP11b在斑马鱼胚胎发育不同时期表达明显,且在耳囊处有持续表达。     

UVA-mediated downregulation of MMP-2 and MMP-9 in human epidermal keratinocytes

While human dermal fibroblasts increase the expression and secretion of distinct matrix metalloproteinases (MMPs) in response to ultraviolet (UV) irradiation, much less is known about regulation of MMPs with regard to normal human epidermal keratinocytes (NHEK). In this in vitro study, the effect of ultraviolet A (UVA) irradiation on gelatinase expression and secretion by NHEK was investigated. Irradiation of NHEK with non-toxic doses of UVA resulted in a dose-dependent downregulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B). A single dose of 30JUVA/cm(2) lowered MMP-2 activity to 26% and MMP-9 activity to 33% compared with mock-irradiated cells at 24h after irradiation. Downregulation of MMP-2 and MMP-9 steady-state mRNA levels was observed at 4h after UVA irradiation. The inhibitory effect of UVA on gelatinases was mediated by UVA-generated singlet oxygen (1O(2)). These findings suggest an inverse response to UVA irradiation in NHEK than in fibroblasts.     

Potential signal pathway of all-trans retinoic acid for MMP-2 and MMP-9 expression in injury podocyte induced by adriamycin

Abstract

All-trans-retinoic acid (ATRA) can regulate some specific genes expression in various tissue and cells via nuclear retinoic acid receptors (RARs), including three subtypes: retinoic acid receptor-alpha (RAR-α), retinoic acid receptor-beta (RAR-β) and retinoic acid receptor-gamma (RAR-γ). Podocyte injury plays a pivotal role in the progression of glomerulosclerosis (GS). This study was performed to study the potential signal pathway of ATRA in the expression of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9) in injury podocyte. Cells were divided into three groups: group of negative control (NC), group of injury podocyte induced by adriamycin (ADR) (AI) and group of ADR inducing podocyte injury model treated with ATRA (AA). The cells morphology changes were detected using microscope and scanning electron microscopy. MMP-2 and MMP-9 enzymic activity was detected using the gelatin zymography method. Protein and mRNA expressions of MMP-2, MMP-9, RAR-α, RAR-β and RAR-γ were measured by western-blot and real-time RT-PCR. Enzymatic activity of MMP-2 and MMP-9 in group AA was significantly enhanced compared to AI group after ATRA-treated 24?h (p?<?0.05). The protein and mRNA expressions of MMP-2/MMP-9 in group AA were significantly increased than those in group AI at both 12 and 24?h time points (p?<?0.05). Compared to group AI, RAR-α and RAR-γ protein/mRNA expressions of group AA were significantly increased at both 12 and 24?h time points (p?<?0.05). There was no difference for the expression of RAR-β between group AI and group AA (p?>?0.05). RAR-α protein level was positively correlated with MMP-2 or MMP-9 protein expression (p?<?0.05), and RAR-γ protein level was also positively correlated with MMP-2 or MMP-9 protein expression (p?<?0.05). In conclusion, ATRA may increase expression of MMP-2 and MMP-9 by the potential signal pathway of RAR-α and RAR-γ in injury podocyte induced by adriamycin, but not RAR-β.     

The role of gelatinases in colorectal cancer progression and metastasis

Various proteases are involved in cancer progression and metastasis. In particular, gelatinases, matrix metalloproteinase-2 (MMP-2) and MMP-9, have been implicated to play a role in colon cancer progression and metastasis in animal models and patients. In the present review, the clinical relevance and the prognostic value of messenger ribonucleic acid (mRNA) and protein expression and proenzyme activation of MMP-2 and MMP-9 are evaluated in relation to colorectal cancer. Expression of tissue inhibitors of MMPs (TIMPs) in relation with MMP expression in cancer tissues and the relevance of detection of plasma or serum levels of MMP-2 and/or MMP-9 and TIMPs for prognosis are also discussed. Furthermore, involvement of MMP-2 and MMP-9 in experimental models of colorectal cancer is reviewed. In vitro studies have suggested that gelatinase is expressed in cancer cells but animal models indicated that gelatinase expression in non-cancer cells in tumors contributes to cancer progression. In fact, interactions between cancer cells and host tissues have been shown to modulate gelatinase expression in host cells. Inhibition of gelatinases by synthetic MMP inhibitors has been considered to be an attractive approach to block cancer progression. However, despite promising results in animal models, clinical trials with MMP inhibitors have been disappointing so far. To obtain more insight in the (patho)physiological functions of gelatinases, regulation of MMP-2 and MMP-9 expression is discussed. Mitogen activated protein kinase (MAPK) signalling has been shown to be involved in regulation of gelatinase expression in both cancer cells and non-cancer cells. Expression can be triggered by a variety of stimuli including growth factors, cytokines and extracellular matrix (ECM) components. On the other hand, MMP-2 and MMP-9 activity regulates bioavailability and activity of growth factors and cytokines, affects the immune response and is involved in angiogenesis. Because of the multifunctionality of gelatinases, it is unpredictable at what stage of cancer development and in which processes gelatinase activity is involved. Therefore, it is concluded that the use of MMP inhibitors to treat cancer should be considered carefully.     

Fine-structural distribution of MMP-2 and MMP-9 activities in the rat skeletal muscle upon training: a study by high-resolution in situ zymography

Matrix metalloproteinases (MMPs) are key regulators of extracellular matrix remodeling, but have also important intracellular targets. The purpose of this study was to examine the activity and subcellular localization of the gelatinases MMP-2 and MMP-9 in skeletal muscle of control and physically trained rats. In control hind limb muscle, the activity of the gelatinases was barely detectable. In contrast, after 5 days of intense exercise, in Soleus (Sol), but not Extensor digitorum longus (EDL) muscle, significant upregulation of gelatinolytic activity in myofibers was observed mainly in the nuclei, as assessed by high resolution in situ zymography. The nuclei of quiescent satellite cells did not contain the activity. Within the myonuclei, the gelatinolytic activity colocalized with an activated RNA Polymerase II. Also in Sol, but not in EDL, there were few foci of mononuclear cells with strongly positive cytoplasm, associated with apparent necrotic myofibers. These cells were identified as activated satellite cells/myoblasts. No extracellular gelatinase activity was observed. Gel zymography combined with subcellular fractionation revealed training-related upregulation of active MMP-2 in the nuclear fraction, and increase of active MMP-9 in the cytoplasmic fraction of Sol. Using RT-PCR, selective increase in MMP-9 mRNA was observed. We conclude that training activates nuclear MMP-2, and increases expression and activity of cytoplasmic MMP-9 in Sol, but not in EDL. Our results suggest that the gelatinases are involved in muscle adaptation to training, and that MMP-2 may play a novel role in myonuclear functions.     

Matrix metalloproteinase 9 (MMP-9) is upregulated during scarless wound healing in athymic nude mice

Cutaneous wound healing is associated with migratory and remodeling events that require the action of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Differences in their expressions were observed during scar-forming and scar-free skin wound healing. We previously found that athymic nude mice are exceptional among mature mammals in their ability to heal injured skin scarlessly. The present study was undertaken to determine whether the modulation of MMP-2 and MMP-9 expression during scarless healing in nude mice was different from scar-forming animals. Full thickness skin wounds were made into the back of nude, wild-type controls (C57BL/6J), immunodeficient SCID and Rag, thymectomized neonates and adults, and cyclosporin A treated mice. Post-injured skin tissues were harvested at Day 7 and 24 after injury. Quantitative RT-PCR, Western blot, gelatin zymography and immunohistochemical assays were performed. Our results show that MMP-2 protein was high but similarly expressed in all post-injured animals on Day 7 after injury. Late phase (Day 24) of wound repair was characterized by a decrease in mRNA and protein expression and a decrease in gelatinolytic activity of MMP-2 in all post-injured samples. On the contrary, high (p < 0.001) levels of mRNA expression, prominent pro-and active forms of MMP-9 and cells immunopositive for MMP-9 were present exclusively in the post-injured tissues from nude mice on Day 24 after wounding. This data suggest that MMP-9 expression in the remodeling phase of wound healing in nude mice could be a major component of their ability for scar-free healing.     

The potential mechanism for the different expressions of gelatinases induced by all-trans retinoic acid in different cells

Gelatinases include matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). The increased expressions of gelatinases are implicated in the pathogenesis of cell injury and cell death. All-trans retinoic acid (ATRA) is an import biological agent which can regulate the expressions of gelatinases and take part in cell injury and cell death. ATRA exerts its biological effect by the high-affinity binding to retinoic acid receptors (RARs). The RARs consist of three isoforms: RAR-α, RAR-β and RAR-γ. However, it is interesting that the effect of ATRA on the expressions of gelatinases is different in different cells. There is no report to explore the possible mechanism for it at present. In this context, we review the published reports and draw a hypothesis that: (i) The distributions of RARs isoforms are different in different cells; (ii) ATRA activates the different RARs isoforms in different cells; (iii) The roles of different RARs isoforms for regulating the expression of MMP-2 or MMP-9 are different in different cells. So, ATRA takes a different function on the expressions of MMP-2 and MMP-9 in different cells. Once the potential strategy can be successfully confirmed, it would be prone to comprehend why the ATRA regulates the different expressions of gelatinases in different cells.     

Expression of gelatinases and their tissue inhibitors in rat corpus luteum during pregnancy and postpartum

Extensive tissue remodeling occurs in the corpus luteum (CL) during both formation and luteolysis. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are believed to play pivotal roles in these processes. In the present study, to evaluate the potential roles of matrix degrading proteases in luteal development and regression, we examined gelatinases and TIMP-1, -2, -3 mRNA expressions, as well as gelatinase activity in rat CL during pregnancy and postpartum using Northern blot, in situ hybridization, and gelatin zymography, respectively. The results showed that MMP-2 mRNA was only expressed at the early stages of pregnancy; TIMP-2 mRNA was highly expressed at the early and late pregnancy and day 1 postpartum, but could not be detected during the mid-phase of pregnancy; TIMP-3 mRNA expression was abundant during early pregnancy and peaked at day 7, but was absent from other time points examined. MMP-9 and TIMP-1 mRNAs in rat CL were below detectable level in the current study. Furthermore, the active MMP-2 was only present during the early stages of pregnancy, and no MMP-9 activity was observed in the zymogram. Taken together, our results suggest that MMP-2 and TIMP-3 may have functional roles in rat luteal formation, while TIMP-2 may be implicated in both formation and regression of the pregnant CL.     

Expression of genes for metalloproteinases (MMP-1, MMP-2, MMP-9, and MMP-12) associated with psoriasis

Using real-time polymerase chain reaction (RT-PCR), we measured mRNA amounts of matrix metalloproteinases (MMPs): MMP-1, MMP-2, MMP-9, and MMP-12 genes in psoriatic lesions and unaffected skin of the same patients. We observed significant (about 15-fold) increase in the expression level of matrix metalloproteinase MMP-1 and MMP-12 genes associated with psoriasis. The results of our studies of MMP gene expression in cultured primary human keratinocytes treated with interleukin (IL-17) have shown upregulation of MMP gene expression both in cultured keratinocytes and in psoriatic skin lesions. Therefore, upregulation of MMP genes in the skin affected by psoriasis could result from IL-17 effects on skin cells.     

非小细胞肺癌组织MMP-2、MMP-9的表达与组织学类型及临床分期的关系

目的:探究非小细胞肺癌组织基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)的表达及其与患者组织学类型及其临床分期的关系。方法:选取2014年1月至2017年1月于我院进行就诊并确诊为非小细胞肺癌的96例患者为实验组,另选取30例肺良性病变患者为对照组,使用免疫组织化学的方法检测患者肺癌组织或肺良性病变组织中MMP-2、MMP-9的表达,并分析MMP-2、MMP-9的表达与患者组织学类型及临床分期之间的关系。结果:非小细胞肺癌组织MMP-2及MMP-9表达水平显著高于肺良性病变组织(P0.05)。非小细胞肺癌鳞癌组织MMP-2、MMP-9表达明显高于腺癌和腺鳞癌(P0.05),而鳞癌与腺鳞癌组织MMP-2、MMP-9表达相比差异无统计学意义(P0.05)。随着非小细胞肺癌临床分期的增加,癌组织MMP-2及MMP-9表达逐渐上升,各分期比较差异均具有统计学意义(P0.05)。结论:MMP-2、MMP-9在非小细胞肺癌组织中的表达水平明显上调,以鳞癌最高,且与临床分期显著相关,提示其对组织学类型、临床分期、病情评估和预后判断均具有一定的参考意义。