Sorsby fundus dystrophy mutation Timp3(S156C) affects the morphological and biochemical phenotype but not metalloproteinase homeostasis

The tissue inhibitor of metalloproteinases-3 (TIMP3) is a multifunctional protein tightly associated with the extracellular matrix (ECM). A specific type of mutation in TIMP3 which results in potentially unpaired cysteine residues at the C-terminus of the protein has been shown to cause Sorsby fundus dystrophy (SFD), an autosomal dominant retinopathy of late onset. An early finding in SFD is a striking accumulation of protein and lipid material in Bruch's membrane, a multilayered ECM structure located between the choroid and the RPE. To study the molecular mechanisms underlying SFD pathology, we recently generated two mouse lines, one deficient in Timp3 (Timp3(-/-)) and one carrying an SFD-related mutation in the orthologous murine Timp3 gene (Timp3(S156C/S156C)). We now established immortalized fibroblast cells from the mutant mouse strains and provide evidence that the various cell lines display distinct morphological and physiological features that are dependent on the mutational status of the Timp3 protein in the secreted ECM. We show that matrix metalloproteinase (MMP) activity and inhibitory properties of Timp3 are not affected by the SFD-associated mutation. We further demonstrate that Timp3(S156C) protein accumulates in the ECM of the mutant fibroblast cells and that this accumulation is not due to a prolonged turnover rate of mutant vs. normal Timp3. We also show that the relative abundance of mutant and normal Timp3 in the ECM has no measurable effects on cellular phenotypes. Together, these findings suggest (i) a functional role of normal Timp3 in pathways determining cellular morphology and (ii) a loss of this particular function as a consequence of the Ser156Cys mutation. We therefore hypothesize that SFD pathogenesis is due to a loss-of-function mutation in TIMP3.     

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.     

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.     

Deregulation of the Egfr/Ras signaling pathway induces age-related brain degeneration in the Drosophila mutant vap

Ras signaling has been shown to play an important role in promoting cell survival in many different tissues. Here we show that upregulation of Ras activity in adult Drosophila neurons induces neuronal cell death, as evident from the phenotype of vacuolar peduncle (vap) mutants defective in the Drosophila RasGAP gene, which encodes a Ras GTPase-activating protein. These mutants show age-related brain degeneration that is dependent on activation of the EGF receptor signaling pathway in adult neurons, leading to autophagic cell death (cell death type 2). These results provide the first evidence for a requirement of Egf receptor activity in differentiated adult Drosophila neurons and show that a delicate balance of Ras activity is essential for the survival of adult neurons.     

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.     

ECM-Regulator timp Is Required for Stem Cell Niche Organization and Cyst Production in the Drosophila Ovary

The extracellular matrix (ECM) is a pivotal component adult tissues and of many tissue-specific stem cell niches. It provides structural support and regulates niche signaling during tissue maintenance and regeneration. In many tissues, ECM remodeling depends on the regulation of MMP (matrix metalloproteinase) activity by inhibitory TIMP (tissue inhibitors of metalloproteinases) proteins. Here, we report that the only Drosophila timp gene is required for maintaining the normal organization and function of the germline stem cell niche in adult females. timp mutant ovaries show reduced levels of both Drosophila Collagen IV α chains. In addition, tissue stiffness and the cellular organization of the ovarian niche are affected in timp mutants. Finally, loss of timp impairs the ability of the germline stem cell niche to generate new cysts. Our results demonstrating a crucial role for timp in tissue organization and gamete production thus provide a link between the regulation of ECM metabolism and tissue homeostasis.     

A mucin-type O-glycosyltransferase modulates cell adhesion during Drosophila development

Cell-cell and cell-matrix adhesion are crucial during many stages of eukaryotic development. Here, we provide the first example that mucin-type O-linked glycosylation is involved in a developmentally regulated cell adhesion event in Drosophila melanogaster. Mutations in one member of the evolutionarily conserved family of enzymes that initiates O-linked glycosylation alter epithelial cell adhesion in the Drosophila wing blade. A transposon insertion mutation in pgant3 or RNA interference to pgant3 resulted in blistered wings, a phenotype characteristic of genes involved in integrin-mediated cell interactions. Expression of wild type pgant3 in the mutant background rescued the wing blistering phenotype, whereas expression of another family member (pgant35A) did not, revealing a unique requirement for pgant3. pgant3 mutants displayed reduced O-glycosylation along the basal surface of larval wing imaginal discs, which was restored with wild type pgant3 expression, suggesting that reduced glycosylation of basal proteins is responsible for disruption of adhesion in the adult wing blade. Glycosylation reactions demonstrated that PGANT3 glycosylates certain extracellular matrix (ECM) proteins. Immunoprecipitation experiments revealed that PGANT3 glycosylates tiggrin, an ECM protein known to bind integrin. We propose that this glycosyltransferase is uniquely responsible for glycosylating tiggrin in the wing disc, thus modulating proper cell adhesion through integrin-ECM interactions. This study provides the first evidence for the role of O-glycosylation in a developmentally regulated, integrin-mediated, cell adhesion event and reveals a novel player in wing blade formation during Drosophila development.     

Curcumin suppresses the expression of extracellular matrix genes in activated hepatic stellate cells by inhibiting gene expression of connective tissue growth factor

Upon liver injury, quiescent hepatic stellate cells (HSCs), the most relevant cell type for hepatic fibrogenesis, become active and overproduce extracellular matrix (ECM). Connective tissue growth factor (CTGF) promotes ECM production. Overexpression of CTGF during hepatic fibrogenesis is induced by transforming growth factor (TGF)-beta. We recently demonstrated that curcumin reduced cell growth and inhibited ECM gene expression in activated HSCs. Curcumin induced gene expression of peroxisome proliferator-activated receptor (PPAR)-gamma and stimulated its activity in activated HSCs, which was required for curcumin to suppress ECM gene expression, including alphaI(I)-collagen. The underlying mechanisms remain largely unknown. The aim of this study was to elucidate the mechanisms by which curcumin suppresses alphaI(I)-collagen gene expression in activated HSCs. We hypothesize that inhibition of alphaI(I)-collagen gene expression in HSCs by curcumin is mediated by suppressing CTGF gene expression through attenuating oxidative stress and interrupting TGF-beta signaling. The present report demonstrated that curcumin significantly reduced the abundance of CTGF in passaged HSCs and suppressed its gene expression. Exogenous CTGF dose dependently abrogated the inhibitory effect of curcumin. Activation of PPAR-gamma by curcumin resulted in the interruption of TGF-beta signaling by suppressing gene expression of TGF-beta receptors, leading to inhibition of CTGF gene expression. The phytochemical showed its potent antioxidant property by significantly increasing the level of total glutathione (GSH) and the ratio of GSH to GSSG in activated HSCs. De novo synthesis of cellular GSH was a prerequisite for curcumin to interrupt TGF-beta signaling and inhibited gene expression of CTGF and alphaI(I)-collagen in activated HSCs. Taken together, our results demonstrate that inhibition of alphaI(I)-collagen gene expression by curcumin in activated HSCs results from suppression of CTGF gene expression through increasing cellular GSH contents and interruption of TGF-beta signaling. These results provide novel insights into the mechanisms underlying inhibition of HSC activation by curcumin.     

Screening for Drosophila proteins with distinct expression patterns during development by use of monoclonal antibodies

Kc 167 is a cell line established from Drosophila embryonic hemocytes and has been shown to express many extracellular matrix (ECM) and other proteins important during development. We have screened monoclonal antibodies (mAbs) raised against heparin affinity purified proteins from conditioned medium of Kc 167 cells to identify novel proteins with important roles for development. One mAb recognized a protein expressed with temporary and tissue specific patterns during Drosophila embryogenesis and larval development. This approach is an alternative to screening of Expression Sequence Tag (EST) clones by in situ hybridization to initiate reverse genetics. In addition, a number of mAbs recognizing ECM proteins were also identified. These mAbs will be useful for biochemical and cell biological analyses of Drosophila ECM proteins.     

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.     

Drosophila caspase DRONC is required for specific developmental cell death pathways and stress-induced apoptosis

Proteases of the caspase family play key roles in the execution of apoptosis. In Drosophila there are seven caspases, but their roles in cell death have not been studied in detail due to a lack of availability of specific mutants. Here, we describe the generation of a specific mutant of the Drosophila gene encoding DRONC, the only caspase recruitment domain (CARD) containing apical caspase in the fly. dronc mutants are pupal lethal and our studies show that DRONC is required for many forms of developmental cell deaths and apoptosis induced by DNA damage. Furthermore, we demonstrate that DRONC is required for the autophagic death of larval salivary glands during metamorphosis, but not for histolysis of larval midguts. Our results indicate that DRONC is involved in specific developmental cell death pathways and that in some tissues, effector caspase activation and cell death can occur independently of DRONC.     

latheo encodes a subunit of the origin recognition complex and disrupts neuronal proliferation and adult olfactory memory when mutant.

The Drosophila latheo (lat) gene was identified in a behavioral screen for olfactory memory mutants. The original hypomorphic latP1 mutant (Boynton and Tully, 1992) shows a structural defect in adult brain. Homozygous lethal lat mutants lack imaginal discs, show little cell proliferation in the CNS of third instar larvae, and die as early pupae. latP1 was cloned, and all of the above mentioned defects of hypomorphic or homozygous lethal lat mutants were rescued with a lat+ transgene. lat encodes a novel protein with homology to a subunit of the origin recognition complex (ORC). Human and Drosophila LAT both associate with ORC2 and are related to yeast ORC3, suggesting that LAT functions in DNA replication during cell proliferation.     

Multilevel regulation of matrix metalloproteinases in tissue homeostasis indicates their molecular specificity in vivo

The matrix metalloproteinases (MMPs) play a crucial role in irreversible remodeling of the extracellular matrix (ECM) in normal homeostasis and pathological states. Accumulating data from various studies strongly suggest that MMPs are tightly regulated, starting from the level of gene expression all the way to zymogen activation and endogenous inhibition, with each level controlled by multiple factors. Recent in vivo findings indicate that cell–ECM and cell–cell interactions, as well as ECM bio-active products, contribute an additional layer of regulation at all levels, indicating that individual MMP expression and activity in vivo are highly coordinated and tissue specific processes.     

The Drosophila melanogaster gene brain tumor negatively regulates cell growth and ribosomal RNA synthesis.

The regulation of ribosome synthesis is likely to play an important role in the regulation of cell growth. Previously, we have shown that the ncl-1 gene in Caenorhabditis elegans functions as an inhibitor of cell growth and ribosome synthesis. We now indicate that the Drosophila melanogaster tumor suppressor brain tumor (brat) is an inhibitor of cell growth and is a functional homolog of the C. elegans gene ncl-1. The brat gene is able to rescue the large nucleolus phenotype of ncl-1 mutants. We also show that brat mutant cells are larger, have larger nucleoli, and have more ribosomal RNA than wild-type cells. Furthermore, brat overexpressing cells contain less ribosomal RNA than control cells. These results suggest that the tumorous phenotype of brat mutants may be due to excess cell growth and ribosome synthesis.