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1.
Gen Comp Endocrinol ; 260: 32-40, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29438675

RESUMO

The study was undertaken to examine mRNA expression and localization of selected matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), and the activity of MMPs in chicken postovulatory follicles (POFs) during their apoptotic regression. Apoptotic cells and apoptosis-related caspase expression and activity were examined as well. Chickens were sacrificed 2 h and 21 h after ovulation, and five POFs (POF1 to POF5) were isolated from the ovaries. It was found that the number of apoptotic cells (TUNEL-positive) increased along with follicle regression. The relative expression (RQ) of caspase-2, -3, -8 and -9 mRNA increased (P < 0.05) in POF5, while the activity of all examined caspases elevated gradually (approximately 80-150%) reaching the highest level in POF3, and then slowly decreased to the value noted in POF1 (P < 0.05 - P < 0.001). Real-time polymerase chain reaction revealed different expression of MMP-2, -7, -9 and TIMP-2 and -3 on mRNA levels, and activity assay showed the changes in activity of MMP-2 and -9 in the POFs. Regression of the follicles was accompanied predominantly by an increase in the relative expression of MMP-2, and a decrease in TIMP-2 and -3 mRNAs (P < 0.05 - P < 0.001). The activity levels of MMP-2 and -9 showed pronounced changes during the examined period. During follicle regression elevated activity of MMP-2 and -9 was found (P < 0.05 - P < 0.001). Immunohistochemistry demonstrated tissue- and follicle-dependent immunoreactivity of the examined members of the MMP system. In summary, the results showing the apoptotic regression-related changes as well as tissue-dependent differences in the expression of selected MMPs and TIMPs, and activity of MMP-2 and MMP-9, point to the significance that these molecules might participate in the complex orchestration of chicken POF regression.


Assuntos
Galinhas/fisiologia , Metaloproteinases da Matriz/fisiologia , Folículo Ovariano/metabolismo , Ovulação/fisiologia , Inibidor Tecidual de Metaloproteinase-2/fisiologia , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Animais , Galinhas/genética , Galinhas/metabolismo , Feminino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/fisiologia , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 7 da Matriz/metabolismo , Metaloproteinase 7 da Matriz/fisiologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/fisiologia , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Ovário/metabolismo , Ovulação/genética , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/metabolismo
2.
Exp Eye Res ; 165: 35-46, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28847738

RESUMO

Sorsby fundus dystrophy (SFD) is an autosomal dominant macular dystrophy with an estimated prevalence of 1 in 220,000 and an onset of disease around the 4th to 6th decade of life. Similar to age-related macular degeneration (AMD), ophthalmoscopy reveals accumulation of protein/lipid deposits under the retinal pigment epithelium (RPE), referred to as drusen, in the eyes of patients with SFD. SFD is caused by variants in the gene for tissue inhibitor of metalloproteinases-3 (TIMP3), which has been found in drusen-like deposits of SFD patients. TIMP3 is constitutively expressed by RPE cells and, in healthy eyes, resides in Bruch's membrane. Most SFD-associated TIMP3 variants involve the gain or loss of a cysteine residue. This suggests the protein aberrantly forms intermolecular disulphide bonds, resulting in the formation of TIMP3 dimers. It has been demonstrated that SFD-associated TIMP3 variants are more resistant to turnover, which is thought to be a result of dimerisation and thought to explain the accumulation of TIMP3 in drusen-like deposits at the level of Bruch's membrane. An important function of TIMP3 within the outer retina is to regulate the thickness of Bruch's membrane. TIMP3 performs this function by inhibiting the activity of matrix metalloproteinases (MMPs), which have the function of catalysing breakdown of the extracellular matrix. TIMP3 has an additional function to inhibit vascular endothelial growth factor (VEGF) signalling and thereby to inhibit angiogenesis. However, it is unclear whether SFD-associated TIMP3 variant proteins retain these functions. In this review, we discuss the current understanding of the potential mechanisms underlying development of SFD and summarise all known SFD-associated TIMP3 variants. Cell culture models provide an invaluable way to study disease and identify potential treatments. These allow a greater understanding of RPE physiology and pathophysiology, including the ability to study the blood-retinal barrier as well as other RPE functions such as phagocytosis of photoreceptor outer segments. This review describes some examples of such recent in vitro studies and how they might provide new insights into degenerative diseases like SFD. Thus far, most studies on SFD have been performed using ARPE-19 cells or other, less suitable, cell-types. Now, induced pluripotent stem cell (iPSC) technologies allow the possibility to non-invasively collect somatic cells, such as dermal fibroblast cells and reprogram those to produce iPSCs. Subsequent differentiation of iPSCs can generate patient-derived RPE cells that carry the same disease-associated variant as RPE cells in the eyes of the patient. Use of these patient-derived RPE cells in novel cell culture systems should increase our understanding of how SFD and similar macular dystrophies develop.


Assuntos
Degeneração Macular , Distrofias Retinianas , Células Cultivadas , Humanos , Degeneração Macular/epidemiologia , Degeneração Macular/etiologia , Degeneração Macular/fisiopatologia , Metaloproteinases da Matriz/fisiologia , Modelos Biológicos , Neovascularização Patológica/fisiopatologia , Distrofias Retinianas/epidemiologia , Distrofias Retinianas/etiologia , Distrofias Retinianas/fisiopatologia , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/fisiologia
3.
Anim Sci J ; 88(9): 1279-1290, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28124491

RESUMO

In vitro maturation (IVM) in serum causes hampered expansion of porcine cumulus-oocyte complexes (COCs) due to excessive alpha2 -macroglobulin (A2M). This study investigated two hypotheses that could explain the effect of A2M: (i) binding of epidermal growth factor (EGF) to A2M, followed by its decreased availability; and (ii) inhibition of zinc-dependent metalloproteases. Cumulus expansion was evaluated based on the diameter of the COCs, the proportion of COCs participating in a floating cloud and the proportion of COCs with loss of cumulus cells. The first hypothesis of decreased EGF availability was tested by increasing the EGF concentration (20 and 50 ng/mL vs. 10 ng/mL), but was not confirmed because cumulus expansion did not improve. To verify the second hypothesis of inhibited zinc-dependent metalloproteases, the effect of tissue inhibitor of metalloproteases-3 (TIMP-3) on cumulus expansion during IVM with and without A2M was investigated. To immuno-neutralize A2M, serum was pre-incubated with A2M antibodies. Impaired cumulus expansion because of TIMP-3 could only be observed during IVM in 10% of serum with A2M antibodies. No effect of TIMP-3 was observed in medium without A2M antibodies. These results indicate that A2M and TIMP-3 share a common target, a zinc-dependent metalloprotease. Future research is directed toward the identification of the protease involved.


Assuntos
Técnicas de Maturação in Vitro de Oócitos , Metaloproteases/antagonistas & inibidores , Oócitos/fisiologia , Zinco , alfa-Macroglobulinas/fisiologia , Animais , Feminino , Suínos , Inibidor Tecidual de Metaloproteinase-3/fisiologia
4.
Circ Res ; 120(1): 49-65, 2017 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-27756793

RESUMO

RATIONALE: Atherosclerosis and aneurysms are leading causes of mortality worldwide. MicroRNAs (miRs) are key determinants of gene and protein expression, and atypical miR expression has been associated with many cardiovascular diseases; although their contributory role to atherosclerotic plaque and abdominal aortic aneurysm stability are poorly understood. OBJECTIVE: To investigate whether miR-181b regulates tissue inhibitor of metalloproteinase-3 expression and affects atherosclerosis and aneurysms. METHODS AND RESULTS: Here, we demonstrate that miR-181b was overexpressed in symptomatic human atherosclerotic plaques and abdominal aortic aneurysms and correlated with decreased expression of predicted miR-181b targets, tissue inhibitor of metalloproteinase-3, and elastin. Using the well-characterized mouse atherosclerosis models of Apoe-/- and Ldlr-/-, we observed that in vivo administration of locked nucleic acid anti-miR-181b retarded both the development and the progression of atherosclerotic plaques. Systemic delivery of anti-miR-181b in angiotensin II-infused Apoe-/- and Ldlr-/- mice attenuated aneurysm formation and progression within the ascending, thoracic, and abdominal aorta. Moreover, miR-181b inhibition greatly increased elastin and collagen expression, promoting a fibrotic response and subsequent stabilization of existing plaques and aneurysms. We determined that miR-181b negatively regulates macrophage tissue inhibitor of metalloproteinase-3 expression and vascular smooth muscle cell elastin production, both important factors in maintaining atherosclerotic plaque and aneurysm stability. Validation studies in Timp3-/- mice confirmed that the beneficial effects afforded by miR-181b inhibition are largely tissue inhibitor of metalloproteinase-3 dependent, while also revealing an additional protective effect through elevating elastin synthesis. CONCLUSIONS: Our findings suggest that the management of miR-181b and its target genes provides therapeutic potential for limiting the progression of atherosclerosis and aneurysms and protecting them from rupture.


Assuntos
Aneurisma da Aorta Abdominal/metabolismo , Aterosclerose/metabolismo , Elastina/fisiologia , MicroRNAs/biossíntese , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Animais , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/prevenção & controle , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Dieta Hiperlipídica/efeitos adversos , Elastina/antagonistas & inibidores , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/antagonistas & inibidores , Pessoa de Meia-Idade , Inibidor Tecidual de Metaloproteinase-3/antagonistas & inibidores
5.
Mol Cell Endocrinol ; 412: 104-15, 2015 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-26054746

RESUMO

Tissue inhibitor of metalloproteinase 3 (TIMP3) played a key role in female reproduction. However, its expression and function in goat are still unclear. In the present study, the full-length cDNA of goat TIMP3 was cloned from adult goat ovary; meanwhile, we demonstrated that putative TIMP3 protein shared a highly conserved amino acid sequence with known mammalian homologs. Real-time PCR results showed that TIMP3 was widely expressed in the tissues of adult goat. In the ovary, increasing expression of TIMP3 mRNA was discovered during the growth process of follicle and corpus luteum. Immunohistochemistry results suggested that TIMP3 protein existed in oocytes of all types of follicles, corpus luteum and granulosa and theca cells of primary, secondary, and antral but not primordial follicles. In vitro, human chorionic gonadotropin (hCG) stimulated the expression of TIMP3 in goat granulosa cells. hCG-induced TIMP3 mRNA expression was reduced by the inhibitors of protein kinase A, protein kinase C, MAPK kinase, or p38 kinase. Functionally, over-expression of TIMP3 significantly increased apoptosis and decreased the viability of cultured granulosa cells. Knockdown of TIMP3 could decrease hCG-induced progesterone secretion and the mRNA abundance of key steroidogenic enzymes (StAR, p450scc and HSD3B) as well as ECM proteins (DCN and FN). These findings provided evidence that the hCG induced expression of TIMP3 may play an important role in regulating goat granulosa cell survival and steroidogenesis.


Assuntos
Expressão Gênica , Cabras/metabolismo , Células da Granulosa/metabolismo , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Sequência de Aminoácidos , Animais , Apoptose , Vias Biossintéticas , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Sequência Conservada , Feminino , Células da Granulosa/efeitos dos fármacos , Dados de Sequência Molecular , Ovário/citologia , Ovário/metabolismo , Progesterona/biossíntese , Transdução de Sinais , Inibidor Tecidual de Metaloproteinase-3/química , Ativação Transcricional
6.
Oncogene ; 34(31): 4098-108, 2015 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-25347747

RESUMO

Inflammation enables human cancers and is a critical promoter of hepatocellular carcinoma (HCC). TIMP3 (Tissue inhibitor of metalloproteinase 3), a natural metalloproteinase inhibitor, controls cytokine and growth factor bioavailability to keep inflammation in check and regulate cell survival in the liver. TIMP3 is also found silenced in human cancers. We therefore tested whether Timp3 affects HCC predisposition. Remarkably, genetic loss of Timp3 protected from carcinogen-induced HCC through the immediate engagement of several tumor suppressor pathways, while tumor necrosis factor (TNF) signaling was dispensable for this protection. All wild-type mice developed HCC by 12 months, whereas HCC incidence was reduced to 33% at 12 months and 57% at 15 months in Timp3 null mice. Upon acute carcinogen treatment the deficient livers exhibited greater cytokine expression, but lower cell death and higher hepatocyte senescence. We found that precocious activation of p53, p38 and Notch preceded senescence and hepatic cell differentiation, and these events were conserved throughout tumorigenesis. Timp3-deficient mouse embryo fibroblasts also responded to carcinogen by favoring senescence over apoptosis. We conclude that Timp3 status determines p53, p38 and Notch coactivation to instruct hepatic cell fate and transformation and uncover mechanisms that are protective even within a pro-inflammatory microenvironment.


Assuntos
Carcinoma Hepatocelular/genética , Resistência à Doença/genética , Neoplasias Hepáticas/genética , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Animais , Apoptose/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/prevenção & controle , Transformação Celular Neoplásica/genética , Células Cultivadas , Senescência Celular/genética , Citoproteção/genética , Embrião de Mamíferos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
7.
Am J Physiol Regul Integr Comp Physiol ; 304(8): R613-20, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23427085

RESUMO

Maternal hypoxia inhibits cardiomyocyte proliferation in the heart of fetal and neonatal rats. The present study tested the hypothesis that hypoxia has a direct effect inhibiting cardiomyocyte proliferation via upregulating tissue inhibitors of metalloproteinases (TIMP) in fetal rat hearts. Isolated fetal rat hearts and rat embryonic ventricular myocyte H9c2 cells were treated ex vivo with 20% or 1% O(2) for 48 or 24 h, respectively. Hypoxia caused a significant reduction in cardiomyocyte Ki-67 expression and bromodeoxyuridine incorporation in fetal hearts and H9c2 cells. In both fetal hearts and H9c2 cells, hypoxia resulted in a significant decrease in a cell division marker cyclin D2 but an increase in a cell division inhibitor p27. Additionally, hypoxia caused an upregulation of TIMP-3 and TIMP-4 in fetal hearts and H9c2 cells. Knockdown of TIMP-3 in H9c2 cells significantly increased cyclin D2 and Ki-67 and partially blocked the hypoxia-induced inhibition of cyclin D2 and Ki-67 in H9c2 cells. Unlike TIMP-3, TIMP-4 knockdown had no significant effects on the basal levels of cell proliferation but completely abrogated the hypoxia-mediated effects. These findings provide evidence of a novel causal role of TIMP-4 and TIMP-3 in the direct inhibitory effect of hypoxia on cardiomyocyte proliferation in the developing heart.


Assuntos
Proliferação de Células , Coração Fetal/fisiologia , Hipóxia Fetal/patologia , Miócitos Cardíacos/fisiologia , Inibidores Teciduais de Metaloproteinases/fisiologia , Animais , Antimetabólitos , Western Blotting , Bromodesoxiuridina , Linhagem Celular , Tamanho Celular , Ciclina D2/biossíntese , Ciclina D2/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Feminino , Imunofluorescência , Antígeno Ki-67/biossíntese , Gravidez , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Inibidores Teciduais de Metaloproteinases/genética , Transfecção , Regulação para Cima/fisiologia , Inibidor Tecidual 4 de Metaloproteinase
8.
Am J Physiol Renal Physiol ; 303(9): F1341-52, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22896043

RESUMO

Diabetic nephropathy is the most common cause of end-stage renal disease. Polymorphism in the tissue inhibitor of metalloproteinase-3 (TIMP3) gene, and the ECM-bound inhibitor of matrix metalloproteinases (MMPs), has been linked to diabetic nephropathy in humans. To elucidate the mechanism, we generated double mutant mice in which the TIMP3 gene was deleted in the genetic diabetic Akita mouse background. The aggravation of diabetic injury occurred in the absence of worsening of hypertension or hyperglycemia. In fact, myocardial TIMP3 levels were not affected in Akita hearts, and cardiac diastolic and systolic function remained unchanged in the double mutant mice. However, TIMP3 levels increased in Akita kidneys and deletion of TIMP3 exacerbated the diabetic renal injury in the Akita mouse, characterized by increased albuminuria, mesangial matrix expansion, and kidney hypertrophy. The progression of diabetic renal injury was accompanied by the upregulation of fibrotic and inflammatory markers, increased production of reactive oxygen species and NADPH oxidase activity, and elevated activity of TNF-α-converting enzyme (TACE) in the TIMP3(-/-)/Akita kidneys. Moreover, while the elevated phospho-Akt (S473 and T308) and phospho-ERK1/2 in the Akita mice was not detected in the TIMP3(-/-)/Akita kidneys, PKCß1 (but not PKCα) was markedly elevated in the double mutant kidneys. Our data provide definitive evidence for a critical and selective role of TIMP3 in diabetic renal injury consistent with gene expression findings from human diabetic kidneys.


Assuntos
Nefropatias Diabéticas/fisiopatologia , Progressão da Doença , Rim/fisiopatologia , Inibidor Tecidual de Metaloproteinase-3/deficiência , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animais , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Modelos Animais de Doenças , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , NADPH Oxidases/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Espécies Reativas de Nitrogênio/metabolismo , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/fisiologia
10.
J Am Soc Nephrol ; 23(5): 868-83, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22383695

RESUMO

Kidney pericytes are progenitors of scar-forming interstitial myofibroblasts that appear after injury. The function of kidney pericytes as microvascular cells and how these cells detach from peritubular capillaries and migrate to the interstitial space, however, are poorly understood. Here, we used an unbiased approach to identify genes in kidney pericytes relevant to detachment and differentiation in response to injury in vivo, with a particular focus on genes regulating proteolytic activity and angiogenesis. Kidney pericytes rapidly activated expression of a disintegrin and metalloprotease with thrombospondin motifs-1 (ADAMTS1) and downregulated its inhibitor, tissue inhibitor of metalloproteinase 3 (TIMP3) in response to injury. Similarly to brain pericytes, kidney pericytes bound to and stabilized capillary tube networks in three-dimensional gels and inhibited metalloproteolytic activity and angiogenic signaling in endothelial cells. In contrast, myofibroblasts did not have these vascular stabilizing functions despite their derivation from kidney pericytes. Pericyte-derived TIMP3 stabilized and ADAMTS1 destabilized the capillary tubular networks. Furthermore, mice deficient in Timp3 had a spontaneous microvascular phenotype in the kidney resulting from overactivated pericytes and were more susceptible to injury-stimulated microvascular rarefaction with an exuberant fibrotic response. Taken together, these data support functions for kidney pericytes in microvascular stability, highlight central roles for regulators of extracellular proteolytic activity in capillary homoeostasis, and identify ADAMTS1 as a marker of activation of kidney pericytes.


Assuntos
Proteínas ADAM/fisiologia , Nefropatias/fisiopatologia , Rim/irrigação sanguínea , Pericitos/fisiologia , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Proteína ADAMTS1 , Animais , Capilares/fisiopatologia , Adesão Celular , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miofibroblastos/fisiologia , Neovascularização Fisiológica , Análise de Sequência com Séries de Oligonucleotídeos , Obstrução Ureteral/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologia
11.
Blood ; 119(20): 4636-44, 2012 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-22415751

RESUMO

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is one of a family of proteins inhibiting matrix metalloproteinases, which has also been identified as a mediator for checking inflammation. Meanwhile, it is well known that inflammation causes the activation of the immune response. However, it is not clear whether TIMP-3 plays a role in the immune system. In the present study, we demonstrated a novel function of TIMP-3 in Th1/Th2 polarization through its influence on the antigen-presenting cells. First, TIMP-3 was found strikingly up-regulated by IL-4 during the differentiation of human dendritic cells via the p38MAPK pathway. Second, the expression of costimulatory molecule-CD86 was repressed by TIMP-3. Besides, the induction of IL-12 in matured dendritic cells was significantly inhibited in a PI3K-dependent manner. Furthermore, dendritic cells matured in the presence of TIMP-3 could stimulate allogeneic naive T helper (Th) cells to display a prominent Th2 polarization. Importantly, in an autoimmune disorder-primary immune thrombocytopenia, TIMP-3 showed a statistically positive correlation with IL-4 and platelet count, but a negative correlation with IFN-γ in patient blood samples. Collectively, these in vitro and in vivo data clearly suggested a novel role of TIMP-3 in Th1/Th2 balance in humans.


Assuntos
Polaridade Celular/genética , Células Dendríticas/fisiologia , Células Th1/fisiologia , Células Th2/fisiologia , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Adolescente , Adulto , Estudos de Casos e Controles , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/imunologia , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/fisiologia , Púrpura Trombocitopênica Idiopática/sangue , Púrpura Trombocitopênica Idiopática/genética , Púrpura Trombocitopênica Idiopática/imunologia , Púrpura Trombocitopênica Idiopática/metabolismo , RNA Interferente Pequeno/farmacologia , Células Th1/citologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th2/citologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Inibidor Tecidual de Metaloproteinase-3/antagonistas & inibidores , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Adulto Jovem
12.
J Clin Invest ; 122(4): 1283-95, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22378045

RESUMO

Malignant gliomas are the most common and the most lethal primary brain tumors in adults. Among malignant gliomas, 60%-80% show loss of P14ARF tumor suppressor activity due to somatic alterations of the INK4A/ARF genetic locus. The tumor suppressor activity of P14ARF is in part a result of its ability to prevent the degradation of P53 by binding to and sequestering HDM2. However, the subsequent finding of P14ARF loss in conjunction with TP53 gene loss in some tumors suggests the protein may have other P53-independent tumor suppressor functions. Here, we report what we believe to be a novel tumor suppressor function for P14ARF as an inhibitor of tumor-induced angiogenesis. We found that P14ARF mediates antiangiogenic effects by upregulating expression of tissue inhibitor of metalloproteinase-3 (TIMP3) in a P53-independent fashion. Mechanistically, this regulation occurred at the gene transcription level and was controlled by HDM2-SP1 interplay, where P14ARF relieved a dominant negative interaction of HDM2 with SP1. P14ARF-induced expression of TIMP3 inhibited endothelial cell migration and vessel formation in response to angiogenic stimuli produced by cancer cells. The discovery of this angiogenesis regulatory pathway may provide new insights into P53-independent P14ARF tumor-suppressive mechanisms that have implications for the development of novel therapies directed at tumors and other diseases characterized by vascular pathology.


Assuntos
Neoplasias Encefálicas/irrigação sanguínea , Regulação Neoplásica da Expressão Gênica , Glioblastoma/irrigação sanguínea , Proteínas de Neoplasias/fisiologia , Neovascularização Patológica/fisiopatologia , Inibidor Tecidual de Metaloproteinase-3/biossíntese , Proteína Supressora de Tumor p14ARF/fisiologia , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular Transformada , Quimiotaxia/efeitos dos fármacos , Neovascularização da Córnea/fisiopatologia , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/patologia , Fibroblastos/efeitos dos fármacos , Genes p53 , Glioblastoma/metabolismo , Humanos , Camundongos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas c-mdm2/fisiologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Fator de Transcrição Sp1/fisiologia , Inibidor Tecidual de Metaloproteinase-3/antagonistas & inibidores , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Transcrição Gênica , Células Tumorais Cultivadas/metabolismo , Regulação para Cima
13.
Diabetes ; 61(2): 454-62, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22228717

RESUMO

The tissue inhibitor of metalloproteinase (TIMP)3, a stromal protein that restrains the activity of proteases and receptors, is reduced in inflammatory metabolic disorders such as type 2 diabetes mellitus (T2DM) and atherosclerosis. We overexpressed Timp3 in mouse macrophages (MacT3) to analyze its potential antidiabetic and antiatherosclerotic effects. Transgenic mice with myeloid cells targeting overexpression of TIMP3 were generated and fed a high-fat diet for 20 weeks. Physical and metabolic phenotypes were determined. Inflammatory markers, lipid accumulation, and insulin sensitivity were measured in white adipose tissue (WAT), liver, and skeletal muscle. In a model of insulin resistance, MacT3 mice were more glucose tolerant and insulin sensitive than wild-type mice in both in vitro and in vivo tests. Molecular and biochemical analyses revealed that increased expression of TIMP3 restrained metabolic inflammation and stress-related pathways, including Jun NH2-terminal kinase and p38 kinase activation, in WAT and liver. TIMP3 overexpression in macrophages resulted in reduced activation of oxidative stress signals related to lipid peroxidation, protein carbonylation, and nitration in WAT and liver. Our data show that macrophage-specific overexpression of TIMP3 protects from metabolic inflammation and related metabolic disorders such as insulin resistance, glucose intolerance, and nonalcoholic steatohepatitis.


Assuntos
Tecido Adiposo/patologia , Fígado Gorduroso/prevenção & controle , Resistência à Insulina , Macrófagos/fisiologia , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Adiposidade , Animais , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Dieta Hiperlipídica , Fluordesoxiglucose F18 , Inflamação/prevenção & controle , Camundongos , Hepatopatia Gordurosa não Alcoólica , Obesidade/metabolismo , RNA Mensageiro/análise , Inibidor Tecidual de Metaloproteinase-3/genética
14.
J Orthop Res ; 30(2): 267-73, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21809381

RESUMO

Intervertebral disc degeneration (IDD) pertains to the loss of extracellular matrix (ECM), particularly the early loss of aggrecan, the turnover of which is regulated by ADAMTSs. Amongst the etiological factors of IDD, mechanical stress plays an important role in the physiological and pathological processes of nucleus pulposus (NP) cells. However, the role of ADAMTSs and their inhibitor in human NP cells under mechanical stress has not been elucidated to date. The purpose of this study was to investigate the role of ADAMTSs and TIMP-3 in NP cells under mechanical stress. Human NP cells isolated from non-degenerative and degenerative discs were subjected to dynamic compressive load. The expression of ADAMTSs, aggrecan, and TIMP-3 was detected by quantitative real-time PCR and/or Western blot. Consequently, the gene expression of ADAMTS-1, 4, and 5 increased significantly in loaded NP cells compared with not-loaded cells from either non-degenerative or degenerative discs, whereas the gene expression of aggrecan decreased significantly. Moreover, Western blot indicated increased protein levels of ADAMTSs-1, 4, and 5. However, the expression of TIMP-3 altered insignificantly. Together, this study is the first addressing the underlying mechanisms of compressive load as a contributing factor to IDD in terms of ADAMTSs. Our results suggest that compressive load leads to the increase in ADAMTS-1, 4, and 5 that contributes to the decrease of aggrecan and IDD via TIMP-3 independent machinery.


Assuntos
Proteínas ADAM/fisiologia , Degeneração do Disco Intervertebral/etiologia , Disco Intervertebral/metabolismo , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Proteínas ADAM/genética , Adolescente , Adulto , Agrecanas/genética , Sobrevivência Celular , Humanos , Disco Intervertebral/citologia , Estresse Mecânico , Inibidor Tecidual de Metaloproteinase-3/genética , Adulto Jovem
15.
J Biol Chem ; 286(49): 42292-42302, 2011 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-22009755

RESUMO

We have shown earlier that miR-221 and -222 are up-regulated in tamoxifen-resistant MCF-7 (OHT(R)) cells and Her2-positive human breast tumors when compared with Her2 negative tumors. In this study, we report markedly enhanced expression of miR-181b in OHT(R) cells and endocrine-resistant tumors. Further, anti-miR-222 or -181b in combination with tamoxifen suppressed growth of tamoxifen-resistant xenografts in mice. Luciferase reporter assay and expression analysis showed that TIMP3, a tissue metalloproteinase inhibitor, is a common target of miR-221/222 and -181b. In situ hybridization and immunohistochemical analysis demonstrated reciprocal relationships between TIMP3 and miR-221/222/181b expression in primary human breast carcinomas. Ectopic expression of TIMP3 inhibited growth of the OHT(R) cells, and its depletion in MCF-7 cells reduced sensitivity to tamoxifen in vitro and in vivo. EGF-induced MAPK and AKT phosphorylation were significantly higher in OHT(R) cells and miR-221/222-overexpressing MCF-7 cells than in control cells, which suggests modulation of mitogenic signaling by TIMP3 and the miRs. On the contrary, phosphoMAPK and phosphoAKT levels were diminished in TIMP3-overexpressing OHT(R) cells and increased in TIMP3-depleted MCF-7 cells. Low levels of estrogen or tamoxifen elicited similar differences in phosphoMAPK levels in these cells. Reduced levels of TIMP3 facilitated growth of tamoxifen-resistant cells by alleviating its inhibitory effect on ADAM10 and ADAM17, which are critical for OHT(R) cell growth. In conclusion, miR-221/222 and -181b facilitate growth factor signaling in tamoxifen-resistant breast cancer by down-regulating TIMP3, and corresponding anti-miRs can be used to render these tumors responsive to tamoxifen.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/metabolismo , MicroRNAs/metabolismo , Tamoxifeno/farmacologia , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação para Baixo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Mamárias Animais/genética , Metaloproteases/metabolismo , Camundongos , Transplante de Neoplasias , Cicatrização
16.
Circulation ; 124(12): 1337-50, 2011 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-21900081

RESUMO

BACKGROUND: Heart failure is one of the leading causes of mortality and is primarily the final stage of several overload cardiomyopathies, preceded by an early adaptive hypertrophic response and characterized by coordinated cardiomyocyte growth, angiogenesis, and inflammation. Therefore, growth factors and cytokines have to be critically regulated during cardiac response to transverse aortic constriction. Interestingly, the dual properties of placental growth factor as an angiogenic factor and cytokine make it a candidate to participate in cardiac remodeling in response to hemodynamic overload. METHODS AND RESULTS: After transverse aortic constriction, placental growth factor knockout mice displayed a dysregulation of cardiac remodeling, negatively affecting muscle growth. Molecular insights underscored that this effect was ascribable mainly to a failure in the establishment of adequate inflammatory response owing to an impaired activity of tumor necrosis factor-α-converting enzyme. Interestingly, after transverse aortic constriction, placental growth factor knockout mice had strongly increased levels of tissue inhibitor of metalloproteinases-3, the main natural TACE inhibitor, thus indicating an unbalance of the tissue inhibitor of metalloproteinases-3/tumor necrosis factor-α-converting enzyme axis. Strikingly, when we used an in vivo RNA interference approach to reduce tissue inhibitor of metalloproteinases-3 levels in placental growth factor knockout mice during transverse aortic constriction, we obtained a complete phenotype rescue of early dilated cardiomyopathy. CONCLUSIONS: Our results demonstrate that placental growth factor finely tunes a balanced regulation of the tissue inhibitor of metalloproteinases-3/tumor necrosis factor-α-converting enzyme axis and the consequent TNF-α activation in response to transverse aortic constriction, thus allowing the establishment of an inflammatory response necessary for adaptive cardiac remodeling.


Assuntos
Proteínas ADAM/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Miocardite/fisiopatologia , Proteínas da Gravidez/fisiologia , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Remodelação Ventricular/fisiologia , Proteínas ADAM/fisiologia , Proteína ADAM17 , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/fisiologia , Animais , Aorta/fisiopatologia , Cardiomiopatia Dilatada/tratamento farmacológico , Cardiomiopatia Dilatada/fisiopatologia , Vasos Coronários/fisiopatologia , Modelos Animais de Doenças , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Masculino , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocardite/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Fator de Crescimento Placentário , Proteínas da Gravidez/genética , Proteínas da Gravidez/farmacologia , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Pressão Ventricular/efeitos dos fármacos , Pressão Ventricular/fisiologia , Remodelação Ventricular/efeitos dos fármacos
17.
Biochem Biophys Res Commun ; 407(2): 277-82, 2011 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-21300026

RESUMO

Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis through the secretion of fibrillar collagens and the tissue inhibitors of metalloproteinase (TIMP)-1 and -2. TIMPs are believed to promote hepatic fibrosis by inhibiting both matrix degradation and apoptosis of HSC. In other cell types, there is evidence that TIMP-1 has effects on proliferation, however the role of TIMPs in the regulation of HSC proliferation remains unexplored. Therefore, we have used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. TIMP-1 and -2 siRNA were highly effective, producing peak target protein knockdown compared to negative control siRNA of 92% and 63%, respectively. Specific silencing of TIMP-1, using siRNA, significantly reduced HSC proliferation. TIMP-1 was localised in part to the HSC nucleus and TIMP-1 siRNA resulted in loss of both cytoplasmic and nuclear TIMP-1. Attenuated proliferation was associated with reduced Akt phosphorylation and was partially rescued by addition of recombinant TIMP-1. We have revealed a novel autocrine mitogenic effect of TIMP-1 on HSC, which may involve Akt-dependent and specific nuclear mechanisms of action. We suggest that TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. Moreover, these findings, together with our previous reports and the emerging data from in vivo studies of TIMP inhibition, provide strong evidence that TIMP-1 is mechanistically central to liver fibrosis and an important potential therapeutic target.


Assuntos
Proliferação de Células , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/genética , RNA Interferente Pequeno/genética , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Animais , Células Cultivadas , Inativação Gênica , Células Estreladas do Fígado/citologia , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/fisiologia , Inibidor Tecidual de Metaloproteinase-3/genética
18.
Placenta ; 32(3): 206-13, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21300402

RESUMO

Abruption-induced thrombin generation and inflammation/infection induced cytokine production have both been associated with fetal membrane (FM) weakening and preterm premature rupture of the fetal membranes (PPROM). Using our in vitro model system we have demonstrated that thrombin, and separately the cytokines, tumor necrosis factor-alpha (TNFα) and interleukin-1-beta (IL-1ß), remodel and weaken full thickness FM. Additionally, we have reported that the anti-oxidant and NFκB inhibitor, alpha-lipoic acid (LA), blocks these thrombin and cytokine induced effects. The purpose of these studies was to determine whether thrombin and cytokines directly weaken the amnion membrane (AM), the major load-bearing component of FM. Isolated AM or full thickness FM fragments from unlabored Cesarean deliveries were incubated with thrombin, TNFα, or IL-1ß, for 48 h. Rupture strength (breaking force) of each fragment was thereafter determined using our published methodology. Biochemical evidence of remodeling and apoptosis; immunoreactive Matrix Metalloproteinase 9 (MMP9), Tissue Inhibitor of Matrix Metalloproteinase 3 (TIMP3) and cleaved poly (ADP-ribose) polymerase (C-PARP) levels in tissue extracts, were determined by western blot and densitometry. Thrombin induced a dose-dependent weakening of isolated AM (P < 0.001) coupled with dose dependent increases in PARP cleavage, and reciprocal increases and decreases, respectively, in MMP9 and TIMP3 protein (all P < 0.01). Thrombin receptor activating peptide-6 (TRAP) also weakened isolated AM. Neither TNFα nor IL-1ß weakened isolated AM. However, both cytokines weakened AM when it was incubated together with the choriodecidua as part of full thickness FM (P < 0.001). Cytokine-conditioned choriodecidua medium also weakened isolated AM (P < 0.001). Under conditions in which cytokines weakened the AM, the changes in MMP9, TIMP3 and PARP cleavage were consistent with those seen after thrombin incubation. LA blocked the FM weakening and remodeling effects. In summary, thrombin weakens AM directly whereas cytokines weaken AM indirectly by causing the release of soluble intermediates from the choriodecidua.


Assuntos
Âmnio/fisiopatologia , Ruptura Prematura de Membranas Fetais/fisiopatologia , Interleucina-1beta/fisiologia , Trombina/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Fosfatase Ácida/farmacologia , Apoptose/fisiologia , Fenômenos Biomecânicos/fisiologia , Western Blotting , Densitometria , Feminino , Humanos , Técnicas In Vitro , Isoenzimas/farmacologia , Metaloproteinase 9 da Matriz/fisiologia , Glicoproteínas de Membrana/fisiologia , Gravidez , Proteínas de Protozoários/fisiologia , Fosfatase Ácida Resistente a Tartarato , Ácido Tióctico/farmacologia , Inibidor Tecidual de Metaloproteinase-3/fisiologia
19.
Oncogene ; 30(27): 3011-23, 2011 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-21339735

RESUMO

Papillary thyroid carcinoma (PTC) arises from the thyroid follicular epithelium and represents the most frequent thyroid malignancy. PTC is associated with gene rearrangements generating RET/PTC and TRK oncogenes, and to the BRAFV600E activating point mutation. A role of tumor-suppressor genes in the pathogenesis of PTC has not been assessed yet. The tissue inhibitor of metalloproteinase-3 (TIMP3) gene, encoding a metalloproteinases inhibitor and capable of inhibiting growth, angiogenesis, invasion and metastasis of several cancers, was found to be silenced by promoter methylation in a consistent fraction of PTCs, in association with tumor aggressiveness and BRAFV600E mutation, thus suggesting an oncosuppressor role. To explore this possibility, in this study we performed gene expression and functional studies. Analysis of gene expression data produced in our laboratory as well as meta-analysis of publicly available data sets confirmed the downregulation of TIMP3 gene expression in PTC with respect to normal thyroid. The functional consequences of TIMP3 downregulation were investigated in the PTC-derived NIM1 cell line, in which the expression of TIMP3 is silenced. Restoration of TIMP3 expression by exposure to soluble TIMP3 protein or by complementary DNA transfection had no effect on the growth rate of NIM1 cells. Instead, it affected the adhesive, migratory and invasive capabilities of NIM1 cells by modulating several proteins involved in these processes. A striking effect was observed in vivo, as TIMP3 reduced the tumorigenicity of NIM1 cells by repressing angiogenesis and macrophage infiltration. Our data indicate that the loss of TIMP3 expression exerts a functional role in the pathogenesis of PTC.


Assuntos
Invasividade Neoplásica/fisiopatologia , Metástase Neoplásica/fisiopatologia , Neoplasias da Glândula Tireoide/patologia , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Metilação de DNA , Humanos , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-3/genética
20.
J Cell Sci ; 123(Pt 17): 2914-21, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20682640

RESUMO

Myogenic differentiation in adult muscle is normally suppressed and can be activated by myogenic cues in a subset of activated satellite cells. The switch mechanism that turns myogenesis on and off is not defined. In the present study, we demonstrate that tissue inhibitor of metalloproteinase 3 (TIMP3), the endogenous inhibitor of TNFalpha-converting enzyme (TACE), acts as an on-off switch for myogenic differentiation by regulating autocrine TNFalpha release. We observed that constitutively expressed TIMP3 is transiently downregulated in the satellite cells of regenerating mouse hindlimb muscles and differentiating C2C12 myoblasts. In C2C12 myoblasts, perturbing TIMP3 downregulation by overexpressing TIMP3 blocks TNFalpha release, p38 MAPK activation, myogenic gene expression and myotube formation. TNFalpha supplementation at a physiological concentration rescues myoblast differentiation. Similarly, in the regenerating soleus, overexpression of TIMP3 impairs release of TNFalpha and myogenic gene expression, and delays the formation of new fibers. In addition, downregulation of TIMP3 is mediated by the myogenesis-promoting microRNA miR-206. Thus, TIMP3 is a physiological regulator of myogenic differentiation.


Assuntos
Desenvolvimento Muscular/fisiologia , Músculo Esquelético/fisiologia , Inibidor Tecidual de Metaloproteinase-3/fisiologia , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animais , Linhagem Celular , Regulação para Baixo , Expressão Gênica , Masculino , Camundongos , MicroRNAs/administração & dosagem , MicroRNAs/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Regeneração/fisiologia , Inibidor Tecidual de Metaloproteinase-3/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
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