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Matrix metalloproteinase (MMP) functions modulate synapse formation and activity-dependent plasticity. Aberrant MMP activity is implicated in fragile X syndrome (FXS), a disease caused by the loss of the RNA-binding protein FMRP and characterized by neurological dysfunction and intellectual disability. Gene expression studies in suggest that Mmps cooperate with the heparan sulfate proteoglycan (HSPG) glypican co-receptor Dally-like protein (Dlp) to restrict trans-synaptic Wnt signaling and that synaptogenic defects in the fly model of FXS are alleviated by either inhibition of Mmp or genetic reduction of Dlp. We used the neuromuscular junction (NMJ) glutamatergic synapse to test activity-dependent Dlp and Mmp intersections in the context of FXS. We found that rapid, activity-dependent synaptic bouton formation depended on secreted Mmp1. Acute neuronal stimulation reduced the abundance of Mmp2 but increased that of both Mmp1 and Dlp, as well as enhanced the colocalization of Dlp and Mmp1 at the synapse. Dlp function promoted Mmp1 abundance, localization, and proteolytic activity around synapses. Dlp glycosaminoglycan (GAG) chains mediated this functional interaction with Mmp1. In the FXS fly model, activity-dependent increases in Mmp1 abundance and activity were lost but were restored by reducing the amount of synaptic Dlp. The data suggest that neuronal activity-induced, HSPG-dependent Mmp regulation drives activity-dependent synaptogenesis and that this is impaired in FXS. Thus, exploring this mechanism further may reveal therapeutic targets that have the potential to restore synaptogenesis in FXS patients.
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Matrix metalloproteinases (MMPs) are extracellular proteases that can cleave extracellular matrix and alter signaling pathways. They have been implicated in many disease states, but it has been difficult to understand the contribution of individual MMPs, as there are over 20 MMPs in vertebrates. The vertebrate MMPs have overlapping substrates, they exhibit genetic redundancy and compensation, and pharmacological inhibitors are non-specific. In contrast, there are only two MMP genes in Drosophila, DmMmp1 and DmMmp2, which makes Drosophila an attractive system to analyze the basis of MMP specificity. Previously, Drosophila MMPs have been categorized by their pericellular localization, as Mmp1 appeared to be secreted and Mmp2 appeared to be membrane-anchored, suggesting that protein localization was the critical distinction in this small MMP family. We report here that products of both genes are found at the cell surface and released into media. Additionally, we show that products of both genes contain GPI-anchors, and unexpectedly, that GPI-anchored MMPs promote cell adhesion when they are rendered inactive. Finally, by using new reagents and assays, we show that the two MMPs cleave different substrates, suggesting that this is the important distinction within this smallest MMP family.
Synaptogenesis requires orchestrated intercellular communication between synaptic partners, with trans-synaptic signals necessarily traversing the extracellular synaptomatrix separating presynaptic and postsynaptic cells. Extracellular matrix metalloproteinases (Mmps) regulated by secreted tissue inhibitors of metalloproteinases (Timps), cleave secreted and membrane-associated targets to sculpt the extracellular environment and modulate intercellular signaling. Here, we test the roles of Mmp at the neuromuscular junction (NMJ) model synapse in the reductionist Drosophila system, which contains just two Mmps (secreted Mmp1 and GPI-anchored Mmp2) and one secreted Timp. We found that all three matrix metalloproteome components co-dependently localize in the synaptomatrix and show that both Mmp1 and Mmp2 independently restrict synapse morphogenesis and functional differentiation. Surprisingly, either dual knockdown or simultaneous inhibition of the two Mmp classes together restores normal synapse development, identifying a reciprocal suppression mechanism. The two Mmp classes co-regulate a Wnt trans-synaptic signaling pathway modulating structural and functional synaptogenesis, including the GPI-anchored heparan sulfate proteoglycan (HSPG) Wnt co-receptor Dally-like protein (Dlp), cognate receptor Frizzled-2 (Frz2) and Wingless (Wg) ligand. Loss of either Mmp1 or Mmp2 reciprocally misregulates Dlp at the synapse, with normal signaling restored by co-removal of both Mmp classes. Correcting Wnt co-receptor Dlp levels in both Mmp mutants prevents structural and functional synaptogenic defects. Taken together, these results identify an Mmp mechanism that fine-tunes HSPG co-receptor function to modulate Wnt signaling to coordinate synapse structural and functional development.
© 2016. Published by The Company of Biologists Ltd.
Breast cancer survival rates decrease from 99% for patients with local disease to 25% for those with distant metastases. Matrix metalloproteinases (MMPs), including MMP2, are associated with metastatic progression. We found that loss of host MMP2 reduces the proliferation of experimental metastases in the lungs and identified fibroblasts in tumour-bearing lungs as the major source of MMP2. In vitro, spheroidal mammary tumour growth was increased by co-culture with control fibroblasts isolated from tumour-bearing lungs, but not when fibroblasts with stable Mmp2 knockdown were used. This result prompted us to assess whether MMP2 was responsible for a tumour-proliferative, activated fibroblast phenotype. To test this, we evaluated: (a) fibroblasts from wild-type tumour-bearing lungs, with or without shRNA-mediated MMP2 knockdown; and (b) normal, quiescent fibroblasts isolated from either WT or Mmp2(-/-) mice. Quantitative PCR revealed that Mmp2 knockdown attenuated expression of two markers of activation (α-smooth muscle actin and vimentin), but there was minimal expression in quiescent WT or Mmp2(-/-) fibroblasts, as expected. Placing quiescent fibroblasts under activating conditions led to increases in activation-associated transcripts in WT but not Mmp2(-/-) fibroblasts. Additionally, Mmp2 knockdown fibroblasts showed significantly decreased expression of the matrix transcripts collagen I, collagen IV and fibronectin. Addition of active TGFβ was sufficient to rescue the MMP2-dependent collagen I and IV expression, while MMP2-induced collagen expression was blocked by the addition of TGFβ1-neutralizing antibody. Gene expression data in stromal cells of human breast cancers reveal that MMP2 expression is also positively correlated with activation and matrix transcripts. Thus, we present a model whereby MMP2 production in tumour fibroblasts is important for TGFβ1 activity and subsequent activation of fibroblasts to a matrix-producing, proliferation-supportive phenotype. Overall, our results reveal a previously undefined role for MMP2 in metastatic outgrowth mediated by fibroblasts, and extend the mechanisms by which MMPs contribute to tumour progression.
Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Ligand-based signaling can potentiate communication between neighboring cells and between cells separated by large distances. In the Drosophila melanogaster ovary, Wingless (Wg) promotes proliferation of follicle stem cells located ~50 µm or five cell diameters away from the Wg source. How Wg traverses this distance is unclear. We find that this long-range signaling requires Division abnormally delayed (Dally)-like (Dlp), a glypican known to extend the range of Wg ligand in the wing disc by binding Wg. Dlp-mediated spreading of Wg to follicle stem cells is opposed by the extracellular protease Mmp2, which cleaved Dlp in cell culture, triggering its relocalization such that Dlp no longer contacted Wg protein. Mmp2-deficient ovaries displayed increased Wg distribution, activity, and stem cell proliferation. Mmp2 protein is expressed in the same cells that produce Wg; thus, niche cells produce both a long-range stem cell proliferation factor and a negative regulator of its spreading. This system could allow for spatial control of Wg signaling to targets at different distances from the source.
© 2014 Wang and Page-McCaw.
Osteoarthritis (OA) of the joint is a prevalent disease accompanied by chronic, debilitating pain. Recent clinical evidence has demonstrated that central sensitization contributes to OA pain. An improved understanding of how OA joint pathology impacts upon the central processing of pain is crucial for the identification of novel analgesic targets/new therapeutic strategies. Inhibitory cannabinoid 2 (CB2) receptors attenuate peripheral immune cell function and modulate central neuro-immune responses in models of neurodegeneration. Systemic administration of the CB2 receptor agonist JWH133 attenuated OA-induced pain behaviour, and the changes in circulating pro- and anti-inflammatory cytokines exhibited in this model. Electrophysiological studies revealed that spinal administration of JWH133 inhibited noxious-evoked responses of spinal neurones in the model of OA pain, but not in control rats, indicating a novel spinal role of this target. We further demonstrate dynamic changes in spinal CB2 receptor mRNA and protein expression in an OA pain model. The expression of CB2 receptor protein by both neurones and microglia in the spinal cord was significantly increased in the model of OA. Hallmarks of central sensitization, significant spinal astrogliosis and increases in activity of metalloproteases MMP-2 and MMP-9 in the spinal cord were evident in the model of OA pain. Systemic administration of JWH133 attenuated these markers of central sensitization, providing a neurobiological basis for analgesic effects of the CB2 receptor in this model of OA pain. Analysis of human spinal cord revealed a negative correlation between spinal cord CB2 receptor mRNA and macroscopic knee chondropathy. These data provide new clinically relevant evidence that joint damage and spinal CB2 receptor expression are correlated combined with converging pre-clinical evidence that activation of CB2 receptors inhibits central sensitization and its contribution to the manifestation of chronic OA pain. These findings suggest that targeting CB2 receptors may have therapeutic potential for treating OA pain.
STUDY QUESTION - Can biologically active vitamin D3 [1,25(OH)₂D3] regulate the expression and activity of matrix metalloproteinases (MMPs) in human uterine fibroid cells?
SUMMARY ANSWER - 1,25(OH)₂D3 effectively reduced the expression and activities of MMP-2 and MMP-9 in cultured human uterine fibroid cells.
WHAT IS KNOWN ALREADY - Uterine fibroids (leiomyoma) express higher levels of MMP activity than adjacent normal myometrium, and this is associated with uterine fibroid pathogenesis. However, it is unknown whether 1,25(OH)₂D3 can regulate the expression and activities of MMPs in human uterine fibroid cells.
STUDY DESIGN, SIZE, DURATION - Surgically removed fresh fibroid tissue was used to generate primary uterine fibroid cells.
PARTICIPANTS/MATERIALS, SETTING, METHODS - An immortalized human uterine fibroid cell line (HuLM) and/or primary human uterine fibroid cells isolated from fresh fibroid tissue were used to examine the expression of several MMPs, tissue inhibitors of metalloproteinases (TIMP) 1 and 2 and the activities of MMP-2 and MMP-9 after 1,25(OH)₂D3 treatment. Real-time PCR and western blots analyses were used to measure mRNA and protein expression of MMPs, respectively. Supernatant cell culture media were analyzed for MMP-2 and MMP-9 activities using a gelatin zymography assay.
MAIN RESULTS AND THE ROLE OF CHANCE - 1-1000 nM 1,25(OH)₂D3 significantly reduced mRNA levels of MMP-2 and MMP-9 in HuLM cells in a concentration-dependent manner (P < 0.5 to P < 0.001). The mRNA levels of MMP-1, MMP-3, MMP-13 and MMP-14 in HuLM cells were also reduced by 1,25(OH)₂D3. 1,25(OH)₂D3 significantly reduced MMP-2 and MMP-9 protein levels in a concentration-dependent manner in both HuLM and primary uterine fibroid cells (P < 0.05 to P < 0.001). Moreover, 1,25(OH)₂D3 increased the mRNA levels of vitamin D receptor (VDR) and TIMP-2 in a concentration-dependent manner in HuLM cells (P < 0.05 to P < 0.01). 1,25(OH)₂D3 also significantly increased protein levels of VDR and TIMP-2 in all cell types tested (P < 0.05 to P < 0.001). Gelatin zymography revealed that pro-MMP-2, active MMP-2 and pro-MMP-9 were down-regulated by 1,25(OH)₂D3 in a concentration-dependent manner; however, the active MMP-9 was undetectable.
LIMITATIONS, REASONS FOR CAUTION - This study was performed using in vitro uterine fibroid cell cultures and the results were extrapolated to in vivo situation of uterine fibroids. Moreover, in this study the interaction of vitamin D3 with other regulators such as steroid hormone receptors was not explored.
WIDER IMPLICATIONS OF THE FINDINGS - This study reveals an important biological function of 1,25(OH)₂D3 in the regulation of expression and activities of MMP-2 and MMP-9. Thus, 1,25(OH)₂D3 might be a potential effective, safe non-surgical treatment option for human uterine fibroids.
During inflammation, vascular permeability is increased by various proteolytic events, such as the generation of bradykinin, that augment local tissue responses by enabling tissue penetration of serum proteins, including complement and acute-phase proteins. Proteases also govern inflammatory responses by processing extracellular matrix proteins and soluble bioactive mediators. We quantified changes in the proteome and the nature of protein amino termini (the N-terminome) and the altered abundance of murine proteases and inhibitors during skin inflammation. Through analysis of the N-terminome by iTRAQ-TAILS, we identified cotranslational and posttranslational αN-acetylation motifs, quantitative increases in protein abundance, and qualitative changes in the proteolytic signature during inflammation. Of the proteins identified in normal skin, about half were cleaved, and phorbol ester-induced inflammation increased the proportion of cleaved proteins, including chemokines and complement proteins, that were processed at previously uncharacterized sites. In response to phorbol ester-induced inflammation, mice deficient in matrix metalloproteinase 2 (MMP2) showed reduced accumulation of serum proteins in the skin and exhibited different proteolytic networks from those of wild-type mice. We found that the complement 1 (C1) inhibitor attenuated the increase in serum protein accumulation in inflamed skin. Cleavage and inactivation of the C1 inhibitor by MMP2 increased complement activation and bradykinin generation in wild-type mice, leading to increased vessel permeability during inflammation, which was diminished in Mmp2(-/-) mice. Thus, our systems-level analysis of proteolysis dissected cleavage events associated with skin inflammation and demonstrated that loss of a single protease could perturb the proteolytic signaling network and enhance inflammation.
Although Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is overexpressed in two-thirds of gastric cancers, its impact on molecular functions has not been fully characterized. In this study, we examined the role of DARPP-32 in gastric cancer cell invasion. Using matrigel-coated Boyden chamber invasion assay, DARPP-32-overexpressing AGS cells showed a three-fold increase in invasion relative to the vector control (P < 0.01). We also tested the transendothelial cell invasion as a measure of cell aggressiveness using the impedance-based human umbilical vein endothelial cells invasion assay and obtained similar results (P < 0.001). Western blot analysis indicated that overexpression of DARPP-32 mediated an increase in the membrane-type 1 matrix metalloproteinase (MT1-MMP) and CXCR4 protein levels. Consistent with the role of MT1-MMP in cleaving extracellular matrix proteins initiating the activation of soluble MMPs, we detected a robust increase in MMP-2 activity in DARPP-32-overexpressing cells. The knockdown of endogenous DARPP-32 in the MKN-45 cells reversed these signaling events and decreased cell invasive activity. We tested whether the invasive activity mediated by DARPP-32 might involve sustained signaling via CXCR4-dependent activation of the MT1-MMP/MMP-2 pathway. The small-molecule CXCR4 antagonist (AMD3100) and CXCR4-siRNA blocked DARPP-32-induced cell invasion. We further examined our hypothesis that DARPP-32 could interact with CXCR4 and stabilize its levels following stimulation with its ligand, CXCL12. Using reciprocal coimmunoprecipitation and immunofluorescence experiments, we found that DARPP-32 and CXCR4 coexist in the same protein complex. DARPP-32 prolonged the CXCR4 protein half-life and reduced ubiquitination of the CXCR4 protein, following treatment with its ligand, CXCL12. In conclusion, these findings show a novel mechanism by which DARPP-32 promotes cell invasion by regulating CXCR4-mediated activation of the MT1-MMP/MMP-2 pathway.
Recently we reported that statins, the competitive inhibitors of the key enzyme regulating the mevalonate pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), decrease proliferation of human endometrial stromal (HES) cells. Furthermore, we found that simvastatin treatment reduces the number and the size of endometrial implants in a nude mouse model of endometriosis. The present study was undertaken to investigate the effect of simvastatin on HES cell invasiveness and on expression of selected genes relevant to invasiveness: matrix metalloproteinase 2 (MMP2), MMP3, tissue inhibitor of matrix metalloproteinase 2 (TIMP2), and CD44. Because statin-induced inhibition of HMGCR reduces the production of substrates for isoprenylation-geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP)-the effects of GGPP and FPP were also evaluated. Simvastatin induced a concentration-dependent reduction of invasiveness of HES cells. This effect of simvastatin was abrogated by GGPP but not by FPP. Simvastatin also reduced the mRNA levels of MMP2, MMP3, and CD44, but increased TIMP2 mRNA; all these effects of simvastatin were partly or entirely reversed in the presence of GGPP. The present findings provide a novel mechanism of action of simvastatin on endometrial stroma that may explain reduction of endometriosis in animal models of this disease. Furthermore, the presently described effects of simvastatin are likely mediated, at least in part, by inhibition of geranylgeranylation.