Molecular dissection of TIMP3 mutation S156C associated with Sorsby fundus dystrophy

Sorsby fundus dystrophy (SFD) is an autosomal dominant macular degeneration of late onset. A key feature of the disease is the thickening of Bruch's membrane, an ECM structure located between the RPE and the choroid. SFD is caused by mutations in the gene encoding the ECM-associated tissue inhibitor of metalloproteases-3 (TIMP3). We have recently generated two Timp3 gene-targeted mouse lines, one deficient for the murine gene (Timp3-/-) and one carrying an SFD-related S156C mutation. Based on extracts and cell cultures derived from tissues of these animals we now evaluated TIMP3 functionality and its contribution to SFD. We show that the activity levels of TIMP3 target proteases including TACE, ADAMTS4/5 and aggrecan-cleaving MMPs are similar in Timp3S156/+ and Timp3S156C/S156C mice when compared to controls. In Timp3-/- mice, a significant enhancement of enzyme activity was observed for TACE but not for ADAMTS4/5 and MMPs indicating a compensatory effect of other inhibitors regulating the latter two groups of proteases. Fibrin bead assays show that angiogenesis in Timp3S156/+ and Timp3S156C/S156C mice is not altered whereas increased formation of capillary tubes was observed in Timp3-/- animals over controls. Rescue experiments using recombinant proteins demonstrate that the inhibitory activities of TIMP3 towards TACE and aggrecan-cleaving MMPs as well as the anti-angiogenic properties of TIMP3 are not impaired by SFD mutation S156C. We finally demonstrate that wild-type and S156C-TIMP3 proteins block the binding of VEGF to its receptor VEGFR2 to a similar extent. Taken together, this study shows that S156C-TIMP3 retains its known functional properties suggesting that causes other than an imbalance in protease or angiogenic activities represent the primary molecular defect underlying SFD.     

Sorsby's fundus dystrophy tissue inhibitor of metalloproteinases-3 (TIMP-3) mutants have unimpaired matrix metalloproteinase inhibitory activities, but affect cell adhesion to the extracellular matrix.

The TIMP family of matrix metalloproteinase inhibitors consists of four members, of which TIMP-1, -2 and -4 are secreted, freely diffusible proteins, whereas TIMP-3 is ECM-associated. Mutations in the TIMP3 gene have been linked to Sorsby's fundus dystrophy (SFD), an autosomal dominant inherited retinal degenerative disease that leads to blindness. The SFD mutations characterized result in introduction of an unpaired cysteine residue in the C-terminal domain of TIMP-3. We have expressed four SFD mutant TIMP-3 proteins in baby hamster kidney (BHK) cells and evaluated their characteristics alongside wild-type TIMP-3. Analysis of the mutant proteins (Ser156Cys, Gly167Cys, Tyr168Cys and Ser181Cys) by SDS-PAGE and reverse zymography revealed that each of the mutants retained gelatinase A and gelatinase B inhibitory activity, and were localized to the ECM. Association rate constants for Ser156Cys TIMP-3 with gelatinase-A, gelatinase-B, stromelysin-1 and collagenase-3 were only moderately reduced compared to wild-type TIMP-3. However, all of the mutants displayed aberrant protein-protein interactions, resulting in the presence of additional proteins or complexes in ECM preparations. Two of the mutants (Ser156Cys and Ser181Cys) showed a marked propensity to form multiple higher molecular-weight complexes that retained TIMP activity on reverse zymography. Expression of the SFD mutant TIMP-3 (and to a lesser extent, wild-type TIMP-3) proteins in BHK cells conferred increased cell adhesiveness to the ECM. Our findings indicate that the pathogenesis of Sorsby's fundus dystrophy cannot be attributed to a failure to localize SFD TIMP-3 proteins to the ECM or defects in MMP inhibition, but may involve the formation of aberrant TIMP-3-containing protein complexes and altered cell adhesion.     

Expression of mutant and wild-type TIMP3 in primary gingival fibroblasts from Sorsby's fundus dystrophy patients

Gingival fibroblast cell lines were derived from Sorsby's fundus dystrophy (SFD) patients carrying the S181C TIMP3 and the E139X TIMP3 mutations. These cell lines were grown in culture to study expression of the wild-type and mutant tissue inhibitor of metalloproteinase 3 (TIMP3) alleles from a normal diploid cell type. Firstly, patient cells were found to co-express the wild-type and mutant TIMP3 alleles, S181C TIMP3 or E139X TIMP3, at the mRNA level using restriction fragment length polymorphism (RFLP) analysis. A SpeI RFLP for E139X TIMP3 is described. Low levels of endogenous TIMP3 protein expression were elevated using the natural polysaccharide calcium pentosan polysulfate (CaPPs) in combination with the cytokine IL-1alpha. Immunoblotting detected protein expression from both wild-type and mutant alleles, S181C TIMP3 or E139X TIMP3. S181C TIMP3 from these cells was found to dimerise and retain MMP2 inhibitory activity. To facilitate studies of the E139X TIMP3 protein, the allele was expressed using HighFive insect cells. In this cell type, the E139X TIMP3 was synthesised as a mixture of monomer and dimer. Both monomeric and dimeric E139X TIMP3 protein retained MMP2 inhibitory activity in gelatin zymography. Expression of mutant E139X or S181C TIMP3 protein from a normal diploid patient-derived fibroblast cell had no effect on either MMP2 or MMP9 expression or activation whilst transcribed from their normal promoter context.     

Expression of Sorsby's fundus dystrophy mutations in human retinal pigment epithelial cells reduces matrix metalloproteinase inhibition and may promote angiogenesis

Sorsby's fundus dystrophy (SFD) is an autosomal dominant degenerative disease of the macula caused by mutations in the tissue inhibitor of metalloproteinase-3 (TIMP-3) gene. Choroidal neovascularization is a hallmark of this disease, which closely resembles the exudative form of age-related macular degeneration. However, the mechanism by which TIMP-3 mutations induce the disease phenotype in SFD remains unknown. To address this question we established human retinal pigment epithelial cell lines expressing wild type or S156C (Ser(156) changed to cysteine) mutant TIMP-3. S156C TIMP-3 had reduced matrix metalloproteinase (MMP) inhibitory activity in retinal pigment epithelial cells and resulted in increased secretion and activation of gelatinase A and B. The conditioned medium from these cells induced angiogenesis in "in vivo" chick chorioallantoic membrane assays that could be reversed with recombinant wild type TIMP-3. Our data indicate that the choroidal neovascularization in SFD may be a result of increased MMP activity, which could lead to the stimulation of angiogenesis. These results also suggest the potential therapeutic use of TIMP-3 or synthetic MMP inhibitors in this disease.     

Gene expression patterns associated with infertility in humans and rodent models

Modern genomic technologies such as DNA arrays provide the means to investigate molecular interactions at an unprecedented level, and arrays have been used to carry out gene expression profiling as a means of identifying candidate genes involved in molecular mechanisms underlying a variety of phenotypes. By comparing gene expression profiles from normal and abnormal human testes with those from comparable infertile mouse models, we endeavored to identify genes and gene networks critical for male fertility. We used commercially available filter-based DNA arrays to analyze testicular gene expression from eight human testis biopsies and three different infertile mouse models (atrichosis mutation, ataxia telangiectasia knockout and CREMtau knockout). Forty-seven mouse genes exhibited differential testicular gene expression (P <0.01) associated with male infertility. These included genes involved in DNA repair (Vim, Rad23A, Rad23B), glutathione metabolism (Gsr, Gstp 1, Mgst1), proteolysis (Ace, Casp1, Ctsd), spermatogenesis (Prlr, Tmsb4 and Zfp-37) and stress response (Hsp 1, Osp94). The expression of 19 human genes was different (P<0.05) between normal and abnormal samples, including those associated with apoptosis (GADD45), gonad development (SOX9), proteolysis (PSMC3, SPINK2, TIMP3, UBE213) and signal transduction (DLK1, NAP4, S100A10). Direct comparison of differentially expressed human and mouse genes identified glucose phosphate isomerase, and the highly similar human tissue inhibitor of metalloproteinase 3 (TIMP3) and mouse Timp2. Using DNA microarrays to profile gene expression in testes from infertile animal models and humans will be useful for understanding congenital infertility, and also infertility caused by environmental exposures where the same genes and molecular mechanisms are involved.     

Loss of Timp3 Gene Leads to Abdominal Aortic Aneurysm Formation in Response to Angiotensin II

Aortic aneurysm is dilation of the aorta primarily due to degradation of the aortic wall extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases (MMPs), the proteases that degrade the ECM. Timp3 is the only ECM-bound Timp, and its levels are altered in the aorta from patients with abdominal aortic aneurysm (AAA). We investigated the causal role of Timp3 in AAA formation. Infusion of angiotensin II (Ang II) using micro-osmotic (Alzet) pumps in Timp3^−/− male mice, but not in wild type control mice, led to adverse remodeling of the abdominal aorta, reduced collagen and elastin proteins but not mRNA, and elevated proteolytic activities, suggesting excess protein degradation within 2 weeks that led to formation of AAA by 4 weeks. Intriguingly, despite early up-regulation of MMP2 in Timp3^−/−Ang II aortas, additional deletion of Mmp2 in these mice (Timp3^−/−/Mmp2^−/−) resulted in exacerbated AAA, compromised survival due to aortic rupture, and inflammation in the abdominal aorta. Reconstitution of WT bone marrow in Timp3^−/−/Mmp2^−/− mice reduced inflammation and prevented AAA in these animals following Ang II infusion. Treatment with a broad spectrum MMP inhibitor (PD166793) prevented the Ang II-induced AAA in Timp3^−/− and Timp3^−/−/Mmp2^−/− mice. Our study demonstrates that the regulatory function of TIMP3 is critical in preventing adverse vascular remodeling and AAA. Hence, replenishing TIMP3, a physiological inhibitor of a number of metalloproteinases, could serve as a therapeutic approach in limiting AAA development or expansion.     

TIMP3 mutation in Sorsby's fundus dystrophy: molecular insights

Sorsby's fundus dystrophy (SFD) is a rare autosomal dominant disorder that results in degeneration of the macular region of the retina, with onset usually in the fourth to fifth decade of life. It leads to the rapid loss of central vision, often followed by further loss of peripheral vision. SFD shares several pathological features commonly found in the 'wet' or exudative form of age-related macular degeneration (AMD), the most common cause of blindness in the elderly in developed countries. These phenotypic similarities have led to SFD being proposed as an acceptable genetic model for AMD. Whereas AMD appears to have a complex aetiology, with both genetic and environmental factors playing a role, SFD has been shown to be a single-gene disorder, linked to mutations in exon 5 of the tissue inhibitor of metalloproteinases 3 (TIMP3) gene on chromosome 22q12-q13. This review confines itself to a discussion of the known biochemical properties of the wild-type and SFD TIMP3 proteins and attempts to relate these to the pathology encountered in SFD patients. We also discuss briefly how, despite the lack of inherited mutations in the structural gene, the TIMP3 protein might play a role in the onset and progression of AMD.     

TIMP3 regulates mammary epithelial apoptosis with immune cell recruitment through differential TNF dependence

Post-lactation mammary involution is a homeostatic process requiring epithelial apoptosis and clearance. Given that the deficiency of the extracellular metalloproteinase inhibitor TIMP3 impacts epithelial apoptosis and heightens inflammatory response, we investigated whether TIMP3 regulates these distinct processes during the phases of mammary gland involution in the mouse. Here we show that TIMP3 deficiency leads to TNF dysregulation, earlier caspase activation and onset of mitochondrial apoptosis. This accelerated first phase of involution includes faster loss of initiating signals (STAT3 activation; TGFβ3) concurrent with immediate luminal deconstruction through E-cadherin fragmentation. Epithelial apoptosis is followed by accelerated adipogenesis and a greater macrophage and T-cell infiltration in Timp3(-/-) involuting glands. Crossing in Tnf deficiency abrogates caspase 3 activation, but heightens macrophage and T-cell influx into Timp3(-/-) glands. The data indicate that TIMP3 differentially impacts apoptosis and inflammatory cell influx, based on involvement of TNF, during the process of mammary involution. An understanding of the molecular factors and wound healing microenvironment of the postpartum mammary gland may have implications for understanding pregnancy-associated breast cancer risk.     

Extracellular matrix regulates endothelial functions through interaction of VEGFR-3 and integrin alpha5beta1

Endothelium extracellular matrix (ECM) interactions can provide distinct spatial and molecular signals which control cellular proliferation, migration, and differentiation. Here, we investigated the role of fibronectin (FN), a major ECM protein, on the functions of lymphatic endothelial cells (LEC). We observed that FN, the ligand for integrin alpha5beta1, selectively promoted the growth of LEC as compared with vitronectin (VN) in the presence of the ligand for vascular endothelial growth factor receptor 3 [VEGFR-3 (VEGF-C156S)]. Upon investigating the mechanisms whereby ECM components regulate VEGFR-3 signaling, we found that FN transactivated VEGFR-3 and significantly enhanced the phosphorylation of VEGFR-3 induced by VEGF-C156S as compared to VN. An enhanced association of the integrin subunit alpha5 or beta1 with VEGFR-3, after stimulation with VEGF-C156S, was observed by co-immunoprecipitation. While blockade of integrin alpha5beta1 inhibited the VEGF-C156S-induced phosphorylation of VEGFR-3, no similar effect was obtained by blocking integrin alphavbeta3. FN also protected the endothelial cells from serum deprivation-induced apoptosis. Moreover, while the specific PI3 kinase inhibitor, LY294002, abolished this FN-mediated cell survival, the MAPK kinase inhibitor, PD98059, had no significant effect. Furthermore, a dominant-negative mutant of VEGFR-3 (G857R) reduced VEGF-C156S or FN-mediated cell survival, as well as the activities of PI3 kinase/Akt. Our results indicate that integrin alpha5beta1 participates in the activation of both VEGFR-3 and its downstream PI3 kinase/Akt signaling pathway, which is essential for FN-mediated lymphatic endothelial cell survival and proliferation.     

人组织型基质金属蛋白酶抑制剂-1在Pichia pastoris酵母中重组表达研究

 为研究组织型基质金属蛋白酶抑制剂 (TIMPs)的分子作用机制 ,探讨了在 Pichia pastoris酵母中高效表达分泌型人组织型基质金属蛋白酶抑制剂 - 1 (TIMP- 1 )的技术路线 ,并对产物性质进行初步研究 .通过 PCR从含有 TIMP- 1基因的 p BS质粒获得了该基因的全长序列 ,构建了 p PIC9/T1表达载体 ,电击法转化酵母 ,通过表型筛选和 PCR鉴定证实了目的基因已稳定整合入 Pichiapastoris酵母基因组中 .SDS- PAGE表明表达量高达 40 mg/L培养上清 .用免疫印迹法确定了产物的正确性 ;同时 ,反向明胶酶谱法证明了重组蛋白具有抑制基质金属蛋白酶的活性 .     

Absence of tissue inhibitor of metalloproteinases 3 disrupts alveologenesis in the mouse

Tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix (ECM) degradation by matrix metalloproteinases (MMPs) throughout lung development. We examined lungs from TIMP3 null mice and found significant air space enlargement compared with wild type (WT) animals during a time course spanning early alveologenesis (post‐partum days 1, 5, 9 and 14). Trichrome staining revealed a similar pattern of collagen distribution in the walls of nascent alveoli; however, the alveolar walls of TIMP3 mutant mice appeared to be thinner than controls. Assessment of MMP2 and MMP9 activities by gelatin zymography demonstrated a significant elevation in the active form of MMP2 at post‐partum days 1 and 5. Treatment of null pregnant dams with a broad spectrum synthetic metalloproteinase inhibitor, GM6001, on embryonic day 16.5 enhanced the formation of primitive alveoli during the saccular stage of lung development as evidenced by a partial, but significant, rescue of alveolar size in post‐partum day 1 animals. We propose that increased MMP activity in the absence of TIMP3 enhances ECM proteolysis, upsetting proper formation of primitive alveolar septa during the saccular stage of alveologenesis. Therefore, TIMP3 indirectly regulates alveolar formation in the mouse. To our knowledge, ours is the first study to demonstrate that in utero manipulation of the TIMP/MMP proteolytic axis, to specifically inhibit proteolysis, significantly affects lung development.     

A novel splice site mutation in the tissue inhibitor of the metalloproteinases-3 gene in Sorsby’s fundus dystrophy with unusual clinical features

Sorsby’s fundus dystrophy (SFD) is an autosomal dominant macular dystrophy which is developed usually in the third or fourth decade of life, and is characterized by central visual loss and nyctalopia due to fundus changes of exudative or atrophic macular lesions. Its functional prognosis is usually poor because of disciform macular scars and peripheral chorioretinal atrophies. To date, five different mutations in the tissue inhibitor of the metallo-proteinases-3 (TIMP3) gene have been identified in families of a wide geographic origin, all of which are missense mutations that cause replacement by cysteine of conserved amino acids in the C-terminus of exon 5 of TIMP3. We have studied two Japanese families with SFD, the first report from the Eastern world, and identified a novel 3’ splice site mutation in the TIMP3 gene, namely a single base insertion at the intron 4/exon 5 junction which converts the consensus sequence CAG to CAAG in the splice acceptor site. In addition, our patients displayed a distinctive clinical expression in that they developed macular dystrophies at an approximately 30-year later age of onset and preserved functional vision until later life with essentially uninvolved peripheral retina. The present findings may provide some insight into the genotype–phenotype relationship in SFD. Received: 27 March 1998 / Accepted: 2 May 1998     

Mice with Tissue Inhibitor of Metalloproteinases 4 (Timp4) Deletion Succumb to Induced Myocardial Infarction but Not to Cardiac Pressure Overload

Tissue inhibitor of metalloproteinases 4 (TIMP4) is expressed highly in heart and found dysregulated in human cardiovascular diseases. It controls extracellular matrix remodeling by inhibiting matrix metalloproteinases (MMPs) and is implicated in processes including cell proliferation, apoptosis, and angiogenesis. Timp4-deficient mice (Timp4^−/−) were generated to assess TIMP4 function in normal development and in models of heart disease. We deleted exons 1–3 of the Timp4 gene by homologous recombination. Timp4^−/− mice are born healthy, develop normally, and produce litters of normal size and gender distribution. These mice show no compensation by overexpression of Timp1, Timp2, or Timp3 in the heart. Following cardiac pressure overload by aortic banding, Timp4^−/− mice have comparable survival rate, cardiac histology, and cardiac function to controls. In this case, Timp4 deficiency is compensated by increased cardiac Timp2 expression. Strikingly, the induction of myocardial infarction (MI) leads to significantly increased mortality in Timp4^−/− mice primarily due to left ventricular rupture. The post-MI mortality of Timp4^−/− mice is reduced by administration of a synthetic MMP inhibitor. Furthermore, combining the genetic deletion of Mmp2 also rescues the higher post-MI mortality of Timp4^−/− mice. Finally, Timp4^−/− mice suffer reduced cardiac function at 20 months of age. Timp4 is not essential for murine development, although its loss moderately compromises cardiac function with aging. Timp4^−/− mice are more susceptible to MI but not to pressure overload, and TIMP4 functions in its capacity as a metalloproteinase inhibitor after myocardial infarction.     

Purified human chondroitin-4-sulfate reduced MMP/TIMP imbalance induced by iron plus ascorbate in human fibroblast cultures

Imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) is an important control point in tissue remodelling. Several findings have reported a marked MMP/TIMP imbalance in a variety of in vitro models in which oxidative stress was induced. Since previous studies showed that commercial hyaluronan and chondroitin-4-sulphate are able to limit lipid peroxidation during oxidative stress, we investigated the antioxidant capacity of purified human plasma chondroitin-4-sulfate in reducing MMP and TIMP imbalance in a model of ROS-induced oxidative injury in fibroblast cultures. Purified human plasma chondroitin-4-sulfate was added to the fibroblast cultures exposed to FeSO4 plus ascorbate. We assayed cell death, MMP and TIMP mRNA expression and protein activities, DNA damage, membrane lipid peroxidation, and aconitase depletion. FeSO4 plus ascorbate produced severe death of cells and increased MMP-1, MMP-2 and MMP-9 expression and protein activities. It also caused DNA strand breaks, enhanced lipid peroxidation and decreased aconitase. TIMP-1 and TIMP-2 protein levels and mRNA expression remain unaltered. Purified human plasma C4S, at three different doses, restored the MMP/TIMP homeostasis, increased cell survival, reduced DNA damage, inhibited lipid peroxidation and limited impairment of aconitase. These results further support the hypothesis that these biomolecules possess antioxidant activity and by reducing ROS production C4S may limit cell injury produced by MMP/TIMP imbalance.     

Physical linkage of the A-raf-1, properdin, synapsin I, and TIMP genes on the human and mouse X chromosomes.

Genes encoding the neuron-specific phosphoprotein synapsin I (SYN1), the glycoprotein tissue inhibitor of metalloproteinases (TIMP), the proto-oncogene A-raf-1 (ARAF1), and properdin (PFC), a positive regulator of the alternative pathway of human complement, lie within a conserved synteny encompassing the proximal short arm of the human X chromosome (Xp21.1-p11) and the centromeric end of the mouse X chromosome (A1-A5). We have used a mouse interspecific cross to demonstrate genetic linkage of Syn-1, Timp, and Araf and also show physical linkage, with Timp lying only 10 kb from Araf, within an intron of the Syn-1 gene. Detailed restriction mapping shows that Timp is transcribed in the same direction as Araf but in the opposite direction to the Syn-1 gene. Analysis of the corresponding region of the human X chromosome indicates a similar arrangement and in addition shows that the properdin gene lies within 5 kb of the 5' end of the synapsin I gene.     

MMP and TIMP temporal gene expression during osteocytogenesis

Osteocytes within bone differentiate from osteoblast precursors which reside in a mineralised extracellular matrix (ECM). Fully differentiated osteocytes are critical for bone development and function but the factors that regulate this differentiation process are unknown. The enzymes primarily responsible for ECM remodelling are matrix metalloproteinases (MMP); however, the expression and role of MMPs during osteocytogenesis is undefined. Here we used MLO-A5 cells to determine the temporal gene expressions of the MMP family and their endogenous inhibitors – tissue inhibitors of metalloproteinases (TIMPs) during osteocytogenesis. RT-qPCR revealed expression of 14 Mmps and 3 Timps in MLO-A5 cells. Mmp2, Mmp23 and Mmp28 were decreased concurrent with mineralisation onset (P < 0.05*). Mmp14 and Mmp19 mRNAs were also significantly increased at day 3 (P < 0.05*) before returning to baseline levels at day 6. Decreased expressions of Timp1, Timp2 and Timp3 mRNA were observed by day 6 compared to day 0 (P < 0.05*). To examine whether these changes are linked to osteocytogenesis, we determined Mmp/Timp mRNA expressions in mineralisation-limited conditions. RT-qPCR revealed that the previously observed decreases in Mmp2, Mmp23 and Mmp28 were not observed in these mineralisation-limited cultures, therefore closely linking these MMPs with osteocyte differentiation. Similarly, we found differential expression of Timp1, Timp2 and Timp3 mRNA in mineralisation-restricted cultures (P < 0.05*). In conclusion, we have identified several members of the MMP/TIMP families as regulators of ECM remodelling necessary for the acquisition of the osteocyte phenotype.