Natural Inhibitors Of Matrix Metalloproteinases Research Articles

Designed Loop Extension Followed by Combinatorial Screening Confers High Specificity to a Broad Matrix Metalloproteinase Inhibitor

Matrix metalloproteinases (MMPs) are key drivers of various diseases, including cancer. Development of probes and drugs capable of selectively inhibiting the individual members of the large MMP family remains a persistent challenge. The inhibitory N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP2), a natural broad MMP inhibitor, can provide a scaffold for protein engineering to create more selective MMP inhibitors. Here, we pursued a unique approach harnessing both computational design and combinatorial screening to confer high binding specificity toward a target MMP in preference to an anti-target MMP. We designed a loop extension of N-TIMP2 to allow new interactions with the non-conserved MMP surface and generated an efficient focused library for yeast surface display, which was then screened for high binding to the target MMP-14 and low binding to anti-target MMP-3. Deep sequencing analysis identified the most promising variants, which were expressed, purified, and tested for selectivity of inhibition. Our best N-TIMP2 variant exhibited 29 pM binding affinity to MMP-14 and 2.4 µM affinity to MMP-3, revealing 7500-fold greater specificity than WT N-TIMP2. High-confidence structural models were obtained by including NGS data in the AlphaFold multiple sequence alignment. The modeling together with experimental mutagenesis validated our design predictions, demonstrating that the loop extension packs tightly against non-conserved residues on MMP-14 and clashes with MMP-3. This study demonstrates how introduction of loop extensions in a manner guided by target protein conservation data and loop design can offer an attractive strategy to achieve specificity in design of protein ligands.

Structure and computation-guided yeast surface display for the evolution of TIMP-based matrix metalloproteinase inhibitors.

The study of protein-protein interactions (PPIs) and the engineering of protein-based inhibitors often employ two distinct strategies. One approach leverages the power of combinatorial libraries, displaying large ensembles of mutant proteins, for example, on the yeast cell surface, to select binders. Another approach harnesses computational modeling, sifting through an astronomically large number of protein sequences and attempting to predict the impact of mutations on PPI binding energy. Individually, each approach has inherent limitations, but when combined, they generate superior outcomes across diverse protein engineering endeavors. This synergistic integration of approaches aids in identifying novel binders and inhibitors, fine-tuning specificity and affinity for known binding partners, and detailed mapping of binding epitopes. It can also provide insight into the specificity profiles of varied PPIs. Here, we outline strategies for directing the evolution of tissue inhibitors of metalloproteinases (TIMPs), which act as natural inhibitors of matrix metalloproteinases (MMPs). We highlight examples wherein design of combinatorial TIMP libraries using structural and computational insights and screening these libraries of variants using yeast surface display (YSD), has successfully optimized for MMP binding and selectivity, and conferred insight into the PPIs involved.

Genetic Variants of Matrix Metalloproteinase and Sepsis: The Need Speed Study.

Many causal mechanisms in sepsis susceptibility are largely unknown and the functional genetic polymorphisms (GP) of matrix metalloproteinases (MMPs) and their natural tissue inhibitor of MMPs (TIMP1) could play a role in its development. GPs of MMPs and TIMP (namely MMP-1 rs1799750, MMP-3 rs3025058, MMP-8 rs11225395, MMP-9 rs2234681, and TIMP-1 rs4898) have been compared in 1058 patients with suspected sepsis to assess the association with susceptibility and etiology of sepsis. Prevalence of MMP8 rs11225395 G/G genotype was higher in sepsis patients than in those with non-infective Systemic Inflammatory Reaction Syndrome (35.6 vs. 26%, hazard ratio, HR 1.56, 95% C.I. 1.04–2.42, p = 0.032). G/G patients developed less hyperthermia (p = 0.041), even after stratification for disease severity (p = 0.003). Patients carrying the 6A allele in MMP3 rs3025058 had a higher probability of microbiologically-proven sepsis (HR 1.4. 95%C.I. 1.01–1.94, p = 0.044), particularly when due to virus (H.R. 2.14, 95% C.I. 1.06–4.31, p = 0.046), while MMP-1 G/G genotype patients carried a higher risk for intracellular bacteria (Chlamydia, Mycoplasma, and Legionella, H.R. 6.46, 95% C.I. 1.58–26.41, p = 0.003). Neither severity of sepsis at presentation, nor 30-day mortality were influenced by the investigated variants or their haplotype. MMP8 rs11225395 G/G carriers have lower temperature at presentation and a more than 50% increased susceptibility to sepsis. Among patients with sepsis, carriers of MMP1 rs1799750 G/G have an increased susceptibility for intracellular pathogen infections, while virus serology is more often positive in those with the MMP3 rs3025058 A/A genotype.

TIMP2 genetic variation rs4789932 may associate with anincreased risk of developing acne scarring based on a case-control study of Chinese Han population.

Acne vulgaris is a common chronic inflammatory cutaneous disorder that has a higher prevalence in adolescents and young adults. Previous studies have indicated that both genetic and environmental factors contribute to its risk. The protein encoded by the TIMP2gene is a natural inhibitor of matrix metalloproteinases (MMPs). Changes in TIMP2 expression are speculated to disrupt the TIMP/MMP balance and result in acne scarring. Our study aimed to comprehensively explore the potential genetic susceptibility of TIMP2 to acne scarring based on a case-control study. In total, 1060 patients with acne scarring (cases) and 2162 patients without acne scarring (controls) were enrolled in the present study. Seventeen tag single-nucleotide polymorphisms (SNPs) in the TIMP2gene were selected for genotyping. Genetic association analyses were conducted at both the single marker and haplotypic levels. Single marker-based association analyses were performed in the genotypic model and allelic model. The distributions of clinical variables in different genotype groups of targeted SNPs in patients with acne scarring were also examined. SNP rs4789932 was identified to be significantly associated with the risk of acne scarring in both the genotypic model (p = 0.001) and allelic model (p = 0.0002). The C allele of SNP rs4789932 was significantly associated with an increased risk of acne scarring (OR [95% CI] = 1.23 [1.10-1.37]). Significant differences were identified between the SNP rs4789932genotypes and the clinical severity of acne scarring (p < 2.2 × 10-16 ). The C allele of SNP rs4789932 was associated with severe clinical features of acne scarring. A significant genetic marker of the promoter region in TIMP2 was identified to contribute to the risk of acne scarring in the Chinese Han population and was significantly associated with the clinical severity of acne scarring in patients.

Engineering Tissue Inhibitors of Metalloproteinases Using Yeast Surface Display.

Yeast surface display (YSD) has been extensively used for protein design, engineering, and directed evolution in the past two decades. Here, we describe methods for directed evolution of tissue inhibitors of metalloproteinase (TIMP), the natural inhibitors of matrix metalloproteinases (MMPs), through design and generation of a combinatorial library of TIMP mutants and screening the targeted TIMP library of variants toward MMP binding using YSD. This protocol can be adopted to other natural enzyme inhibitors and similar protein binders such as antibodies.

Bonding performance of self adhesive flowable composite to dentin pretreated with four different natural matrix metalloproteinases inhibitors

Bonding performance of self adhesive flowable composite to dentin pretreated with four different natural matrix metalloproteinases inhibitors

Matrix metalloproteinase contribution in management of cancer proliferation, metastasis and drug targeting.

Matrix metalloproteinases (MMPs) or matrixins, are members of a zinc-dependent endopeptidase family. They cause remodeling of the extracellular matrix (ECM) leading to numerous diseases. MMPs subfamilies possess: collagenases, gelatinases, stromelysins and membrane-type MMPs (MT-MMP). They consist of several domains; pro-peptide, catalytic, linker peptide and the hemopexin (Hpx) domains. MMPs are involved in initiation, proliferation and metastasis of cancer through the breakdown of ECM physical barriers. Overexpression of MMPs is associated with poor prognosis of cancer. This review will discuss both types of MMPs and current inhibitors, which target them in different aspects, including, biosynthesis, activation, secretion and catalytic activity. Several synthetic and natural inhibitors of MMPs (MMPIs) that can bind the catalytic domain of MMPs have been designed including; peptidomimetic, non-peptidomimetic, tetracycline derivatives, off-target MMPI, natural products, microRNAs and monoclonal antibodies.

Antiphotoaging Effects of 3,5-Dicaffeoyl-epi-quinic Acid via Inhibition of Matrix Metalloproteinases in UVB-Irradiated Human Keratinocytes.

UVB exposure is one of the causes of several skin complications including but not limited to premature aging, wrinkle formation, and hyperpigmentation. UV-induced skin aging is called photoaging, and oxidative stress-induced overexpression of matrix metalloproteinases (MMPs) is the main reason behind the photoaging-mediated collagen degradation. Natural origin inhibitors of MMPs are regarded as a promising approach to prevent or treat photoaging. Therefore, the present study investigated the protective effects of 3,5-dicaffeoyl-epi-quinic acid (DCEQA) in human HaCaT keratinocytes against UVB irradiation-related dysregulation of MMPs. Changes in the mRNA and protein expression and release of MMP-1, -2, and -9 were observed after UVB irradiation with or without DCEQA treatment. In addition, the effect of DCEQA on the activation of p38, JNK, and ERK MAPKs was analyzed. Treatment of UVB-irradiated HaCaT cells with 10 μM DCEQA significantly suppressed the overexpression of both mRNA and protein of MMP-1, -2, and -9 while slightly increasing the diminished type I procollagen production. UVB-induced activation of MAPKs was also ameliorated by DCEQA treatment in a dose-dependent manner. Results indicated that DCEQA treatment was able to protect keratinocytes from UVB-induced photoaging by inhibiting the stimulated production of MMPs and the related decrease in collagen production. It was suggested that DCEQA downregulated the collagen degradation via inhibition of MAPK activation, which resulted in decreased MMP activity.

Directed evolution of the metalloproteinase inhibitor TIMP-1 reveals that its N- and C-terminal domains cooperate in matrix metalloproteinase recognition

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs), enzymes that contribute to cancer and many inflammatory and degenerative diseases. The TIMP N-terminal domain binds and inhibits an MMP catalytic domain, but the role of the TIMP C-terminal domain in MMP inhibition is poorly understood. Here, we employed yeast surface display for directed evolution of full-length human TIMP-1 to develop MMP-3-targeting ultrabinders. By simultaneously incorporating diversity into both domains, we identified TIMP-1 variants that were up to 10-fold improved in binding MMP-3 compared with WT TIMP-1, with inhibition constants (Ki ) in the low picomolar range. Analysis of individual and paired mutations from the selected TIMP-1 variants revealed cooperative effects between distant residues located on the N- and C-terminal TIMP domains, positioned on opposite sides of the interaction interface with MMP-3. Crystal structures of MMP-3 complexes with TIMP-1 variants revealed conformational changes in TIMP-1 near the cooperative mutation sites. Affinity was strengthened by cinching of a reciprocal "tyrosine clasp" formed between the N-terminal domain of TIMP-1 and proximal MMP-3 interface and by changes in secondary structure within the TIMP-1 C-terminal domain that stabilize interdomain interactions and improve complementarity to MMP-3. Our protein engineering and structural studies provide critical insight into the cooperative function of TIMP domains and the significance of peripheral TIMP epitopes in MMP recognition. Our findings suggest new strategies to engineer TIMP proteins for therapeutic applications, and our directed evolution approach may also enable exploration of functional domain interactions in other protein systems.

Structural Elucidation of Engineered Tissue Inhibitor of Metalloproteinases‐1 (TIMP‐1) Variants with Improved Binding Affinity toward Matrix Metalloproteinase‐3 (MMP‐3)

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs), enzymes known for contributions to diseases including cancer and fibrosis. The TIMP N‐terminal domain, regarded as the “inhibitory domain”, has been the center of attention both for understanding natural regulation of MMPs by TIMPs, and for developing protein‐based MMP inhibitors for therapeutic applications. The role of the TIMP C‐terminal domain in MMP inhibition has not been well studied. I have used directed evolution via yeast surface display for protein engineering of full‐length human TIMP‐1 toward development of MMP‐3‐targeted ultra‐binders. TIMP‐1 variants with up to 10‐fold improvement in MMP‐3 binding (compared to WT‐TIMP‐1) were isolated after six rounds of library screening using fluorescence‐activated cell sorting (FACS). Analysis of individual and paired mutations from the selected TIMP‐1 variants revealed a cooperative effect between distant mutations on the N‐ and C‐terminal TIMP domains, positioned on opposite sides of the interaction interface with MMP‐3. I have co‐crystallized two TIMP‐1 variants containing single and composite mutations in complex with the catalytic domain of MMP‐3 and solved the X‐ray crystal structures of these complexes. The protein structures reveal substantial conformational changes in the TIMP‐1 binding interfaces near the two cooperative mutations. Affinity appears to be strengthened by tighter cinching of a reciprocal tyrosine clasp formed between the N‐terminal loop of TIMP‐1 and the proximal MMP3 interface, and adoption of an alpha‐helical conformation by a segment of the TIMP‐1 C‐terminal domain, resulting in stabilized interactions between the two TIMP‐1 domains and improved hydrophobic interactions between TIMP‐1 and MMP‐3. Our protein engineering and structural studies provide novel insight into the cooperative function of N‐ and C‐terminal TIMP domains and the significance of peripheral TIMP epitopes in MMP recognition. Our results further point to new approaches for design of TIMP‐based protein therapeutics with improved affinity and selectivity.Support or Funding InformationThis work was supported by grants DOD W81XWH‐16‐2‐0030, and NIH R21 CA205471. Diffraction data were measured at the Berkeley National Synchrotron Light Source.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Engineering Tissue Inhibitor of Metalloproteinases‐1 (TIMP‐1) as a Selective Inhibitor of Matrix Metalloproteinase‐3 (MMP‐3) for Therapeutic Targeting

Matrix metalloproteinases (MMPs) play a pivotal role in progression of several diseases including cancer and fibrosis, which makes these enzymes promising targets for developing protein therapeutics. The challenge in using MMP inhibitors as drugs in MMP‐associated diseases is lack of high selectivity toward specific target MMPs. Therefore, development of selective MMP inhibitors is a critical goal. Tissue inhibitors of metalloproteinases (TIMPs), as natural MMP inhibitors, offer ideal scaffolds for engineering highly selective MMP‐targeted therapeutics. We have previously shown a key role for MMP‐3 in the pathogenesis of pulmonary fibrosis. TIMP‐1 is a natural MMP‐3 inhibitor with picomolar affinity; it also binds to other MMPs with a spectrum of affinities. Engineering of TIMP‐1 for more selective inhibition of MMP‐3 may provide protein therapeutics with applications in treating lung fibrosis.I have used directed evolution and yeast surface display toward development of selective MMP‐3‐targeted biologic agents through protein engineering of TIMP‐1. I have screened a library of TIMP‐1 mutants generated by random mutagenesis of residues comprising the MMP‐interacting loops of TIMP‐1 to select for enhanced MMP‐3 binding on the yeast surface. Diversified residues have been chosen based on structural studies of TIMP‐1/MMP‐3 interactions. TIMP‐1 mutants isolated after rounds of Fluorescent‐Activated Cell Sorting (FACS) showed substantial improvement in MMP‐3 binding compared to the wild‐type TIMP‐1, both in the surface display context and in enzyme inhibition studies using soluble purified proteins.I have also developed a counter‐selection strategy to screen TIMP‐1 mutants that bind selectively to MMP‐3 in the presence of MMP‐10, an MMP with the greatest sequence and structural similarity to MMP‐3. The isolated TIMP‐1 mutants after five rounds of competitive screening showed up to 20‐fold improvement in binding selectivity, highlighting the significant potential of this approach for development of inhibitors with single‐MMP selectivity. In ongoing studies, I have identified some of the key TIMP‐1 mutations responsible for improvements in MMP‐3 affinity and selectivity. I have co‐crystallized one of the top affinity mutants with the catalytic domain of MMP‐3, and have obtained synchrotron diffraction data at a resolution of 2.2 Å; efforts to solve this crystal structure are underway. I anticipate that this structure will offer insights into the structural basis for improvements in selectivity, and will help to guide future library design for refinement of my directed evolution methodology for generating selective engineered TIMPs. This work provides a promising and innovative strategy to develop protein therapeutics based on natural enzyme inhibitors, critically improving selectivity to eliminate offtarget effects.Support or Funding InformationThis project is funded through two grants from Department of Defense (DoD) (ID # W81XWH‐16‐2‐003015), and National Institute of Health (NIH) (R21CA205471).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of C-reactive protein on the expression of matrix metalloproteinases and their inhibitors via Fcγ receptors on 3T3-L1 adipocytes.

The association between obesity and inflammation is well documented in epidemiological studies. Proteolysis of extracellular matrix (ECM) proteins is involved in adipose tissue enlargement, and matrix metalloproteinases (MMPs) collectively cleave all ECM proteins. Here, we examined the effects of C-reactive protein (CRP), an inflammatory biomarker, on the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs), which are natural inhibitors of MMPs, in adipocyte-differentiated 3T3-L1 cells. We analyzed the expression of Fcγ receptor (FcγR) IIb and FcγRIII, which are candidates for CRP receptors, and the effects of anti-CD16/CD32 antibodies, which can act as FcγRII and FcγRIII blockers on CRP-induced alteration of MMP and TIMP expression. Moreover, we examined the effects of CRP on the activation of mitogen-activated protein kinase (MAPK) signaling, which is involved in MMP and TIMP expression, in the presence or absence of anti-CD16/CD32 antibodies. Stimulation with CRP increased MMP-1, MMP-3, MMP-9, MMP-11, MMP-14, and TIMP-1 expression but did not affect MMP-2, TIMP-2, and TIMP-4 expression; TIMP-3 expression was not detected. Adipocyte-differentiated 3T3-L1cells expressed FcγRIIb and FcγRIII; this expression was upregulated on stimulation with CRP. Anti-CD16/CD32 antibodies inhibited CRP-induced expression of MMPs, except MMP-11, and TIMP-1. CRP induced the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and p38 MAPK but did not affect SAPK/JNK phosphorylation, and Anti-CD16/CD32 attenuated the CRP-induced phosphorylation of p38 MAPK, but not that of ERK1/2. These results suggest that CRP facilitates ECM turnover in adipose tissue by increasing the production of multiple MMPs and TIMP-1 in adipocytes. Moreover, FcγRIIb and FcγRIII are involved in the CRP-induced expression of MMPs and TIMP-1 and the CRP-induced phosphorylation of p38, whereas the FcγR-independent pathway may regulate the CRP-induced MMP-11 expression and the CRP-induced ERK1/2 phosphorylation.

Matrix metalloproteinase protein inhibitors: highlighting a new beginning for metalloproteinases in medicine

Matrix metalloproteinase protein inhibitors: highlighting a new beginning for metalloproteinases in medicine Barbara J Boucher The Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UKThe review by Mohan et al1 is interesting because increased activity of various matrix metalloproteinases (MMPs), increasing the severity of tissue damage, is found in many different disease processes. To reduce tissue-damaging overactivity of these tissue remodeling enzymes, one can reduce MMP secretion, or increase the secretion of tissue inhibitors of MMPs (TIMPs); developing therapeutic agents, or boosting natural tissue MMP inhibitors, is therefore an important and worthwhile project. However, this review does not mention vitamin D which is essential for bone health and is known to regulate bone growth through MMP modulation,2 and increasingly reported as suppressing the overactivity of certain MMPs, especially MMP-2 and MMP-9, in many pathological disorders (although vitamin A can also modulate some MMPs). View the original paper by Mohan and colleagues.

Matrix Metalloproteinases: New Targets in Cancer Therapy

Matrix metalloproteinases (MMPs), the entities responsible for eradicating the structure of extracellular matrix - ECM, are the zinc or calcium ion-dependent enzymes. This family of enzymes embodies a vast spectrum of proteases ranging from collagenases, stromelysins to the membrane type MMPs. The tasks linked with these enzymes are significant not only for a sound and stable development of the body but are also found guilty of carrying out angiogenesis, promoting tumor development and thus providing means to disseminate the cancer cells to the sites other than the primary tumor locale. At each step of angiogenesis, MMPs are operating, thereby featuring endothelial cells and several growth factors like VEGF, FGF, etc. Activation of pro-MMP2 with the involvement of MT1-MMP is one of the key steps that lead to the synthesis of blood vessels from an already existing one. For limiting the action of MMPs, various therapeutic techniques highlighting the mechanism of MMP inhibition have been studied. Several agents have been investigated for phase I, II and III clinical trials in combination with other anti-cancer therapies. Natural endogenous inhibitors of MMPs, TIMPs have a limited half-life and are thus not suitable for the desired outcome. Synthetic agents like Marimastat and BMS-275291 have shown reliable results. Nonetheless, explicit research is required for novel agents being designed and synthesized to attenuate the activity of matrix metalloproteinases that are accountable for cancer metastasis.

TIMP-1 inhibition of occludin degradation in Caco-2 intestinal cells: a potential protective role in necrotizing enterocolitis.

Necrotizing enterocolitis (NEC), a common intestinal disease affecting premature infants, is a major cause of morbidity and mortality. Previous reports indicate an upregulation of intestinal matrix metalloproteinases (MMPs) activity that may play key roles on the higher permeability of the intestinal barrier, typical to NEC. Recently, TIMP-1, a natural inhibitor of MMP's, was found to be over expressed in preterm human breast milk (HBM). Previous studies have shown that infants fed with HBM have a significant reduction in the incidence of NEC. The aim of the present study was to investigate the possible role that TIMP-1 may play on the maintenance of tight junctions and therefore the gut barrier integrity. Timp-1-treated Caco-2 intestinal cells were tested for MMP-2 enzymatic activity and cell junction integrity. TIMP-1 inhibited MMP-2 activity, which induced a significant increase in the expression of occludin but not of claudin-4. TIMP-1 did not affect apoptosis. One of the putative mechanisms associated with HBM protection against NEC is mediated by TIMP-1, which downregulates MMP-2 activity, inhibits the degradation of occluding, and preserves tight junctions and gut barrier integrity.

Effect of Taurine on In Vitro Migration of MCF-7 and MDA-MB-231 Human Breast Carcinoma Cells.

Breast cancer is the most frequent cause of cancer-related deaths in women. Large number of scientists report that 17β-estradiol promotes breast cancer cell proliferation, motility and invasion. However the detailed mechanism remains largely unknown. Metallopeptidase inhibitors, tissue inhibitors of metalloproteinases (TIMPs), are glycoproteins that are expressed in several tissues of organisms. The glycoproteins are natural inhibitors of matrix metalloproteinases (MMPs), a group of peptidases implicated in degradation of the extracellular matrix. In this study we investigated the effect of taurine on migration-related genes expressions in human breast cancer cells. Our results showed that the expressions of migration-related genes such as VEGF and MMPs were significantly stimulated by 17β-estradiol. On the contrary, taurine significantly blocked the expression of VEGF and MMPs against 17β-estradiol. In addition, taurine increased the expression of TIMPs which were suppressed by 17β-estradiol. We also demonstrated that taurine suppressed cell migration in a dose-dependent manner using Radius™ technology.

Interaction of ERK1/2 and Smad2/3 signaling pathways in TGF-β1-induced TIMP-3 expression in rat chondrocytes

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is an important natural inhibitor of matrix metalloproteinases (MMPs) and of a disintegrin and metalloproteinase with thrombospondin motif (ADAMTs), which can cleave cartilage extracellular matrix components to cause cartilage degradation. In this study, our data suggest TGF-β1 induces TIMP-3 expression through activations of both the ERK1/2 and Smad2/3 signaling pathways. TGF-β1-stimulated TIMP-3 expression was significantly inhibited by SB525334 (TGF-β receptor I kinase inhibitor), accompanied by a reduction in ERK1/2 and Smad3 phosphorylation. We used PD98059 (MEK inhibitor) and SIS3 (inhibitor of Smad3 phosphorylation) to investigate the respective roles of ERK1/2 and Smad2/3 signaling pathways in TGF-β1-induced TIMP-3 expression. The results show PD98059 treatment significantly suppressed TGF-β1-induced ERK1/2 phosphorylation and TIMP-3 expression. Under these conditions, the degree of Smad3 phosphorylation correlated with ERK1/2 activation, which suggests that ERK1/2 may activate Smad3 phosphorylation. SIS3 significantly inhibited TGF-β1-induced Smad3 phosphorylation and TIMP-3 expression. ERK1/2 phosphorylation alone had no effect on TGF-β1-induced TIMP-3 expression, which suggests ERK1/2 via Smad3 phosphorylation regulates TGF-β1-induced TIMP-3 expression. Here, we demonstrate that ERK1/2 may be capable of activating the Smad2/3 signaling pathway to result in TGF-β1-induced TIMP-3 up-regulation.

English

Tumor invasion and metastasis are the main causes of cancer death. Matrix metalloproteinases (MMPs) are endopeptidases which degredate the extracellular matrix proteins. MMPs have an important role in tumor progression and metastasis.&nbsp;Fibrosarcoma has been recognized as a highly metastatic cancer which its current therapeutic methods have not been very successful. Medicinal plants are widely used in treatment of cancers. Spearmint is a herb with well known anti-tumor activities. In the present study, the effect of hydro extract of spearmint on MMP-2/9 activities in Wehi-164 fibrosarcoma cells has been evaluated&nbsp;in vitro. Wehi-164 cells were cultured&nbsp;in Roswell Park Memorial Institute (RPMI) medium with 10% fetal bovine serum (FBS). Then the cells at logarithmic growth phase were incubated with different concentrations of hydro extract of spearmint (0.1 to 10 mg/ml) in the presence or absence of&nbsp;phorbol myristate acetate (PMA) (25 ng/ml) for 24, 48 and 72 h. The MMP-2/MMP-9 activity in conditioned medium was evaluated by gelatin zymography.Spearmint aqueous extract significantly reduced the MMP-2 and PMA-induced MMP-9 activity in fibrosarcoma Wehi-164 cells dose dependently. The aqueous extract of spearmint showed inhibitory effect on MMP-2/MMP-9 activity in Wehi-164 fibrosarcoma cells. So, the anti-tumor effects of spearmint may be in part due to its inhibitory effects on MMP-2/MMP-9 activity. Evaluation of effective component (s) in the spearmint extract with anti-MMP activity and their mechanism of action could be useful in designing novel natural MMP inhibitors. &nbsp; Key words:&nbsp;Spearmint, matrix metalloproteinases, fibrosarcoma.

Protein Kinase C-Mediated Regulation of Matrix Metalloproteinase and Tissue Inhibitor of Metalloproteinase Production in a Human Retinal Müller Cells

Purpose: Matrix metalloproteinases (MMPs) play an important role in the degradation of extracellular matrix (ECM) proteins in the retina. Breakdown of ECM proteins results in neovascularization and tractional retinal detachment, which eventually lead to the symptoms of proliferative diabetic retinopathy. Müller cells are reported to be one of the MMP-producing cells in the retina. However, the molecular mechanism of MMP production derived from Müller cells remains to be fully elucidated.Materials and Methods: The human retinal Müller cell line (MIO-M1) was continuously-subcultured in high glucose (25 mM) condition. After the cells reached confluence, they were treated for 24 h with phorbol ester and/or a protein kinase C (PKC) inhibitor, GF109203X in high (25 mM) or low (5 mM) glucose condition. Gelatinase activities in conditioned medium were assessed using gelatin zymography. RT-PCR was performed to analyze the mRNA expression level of MMP-9. Western blot analysis used to detect the protein expression of tissue inhibitors of metalloproteinases (TIMPs). Electrophoresis mobility shift assay was conducted to examine transcription factors involved in MMP-9 transcription.Results: We demonstrated the protein kinase C (PKC)-mediated regulation of proMMP-9 transcription and protein expression through the action of phorbol ester, an activator of PKC. Moreover, we demonstrated the expression of TIMPs, known as natural inhibitors of MMPs at the protein level in a human retinal Müller cell line for the first time, and report that production of these proteins was also regulated in a PKC-dependent manner.Conclusion: Our results suggest that imbalance between MMP and TIMP proteins may promote neovascularization by PKC activation in retinal Müller cells. In addition, the development of novel compounds with regulatory action on MMP and TIMP production through inhibiting PKC activity in retinal Müller cells may lead to new therapeutic approaches for the treatment and prevention of diabetic retinopathy.

Abstract 5294: Increased angiogenesis resulting from TIMP-1 inhibition of TSP-1 is mediated by PI3kinase and FAK interactions

Abstract Tissue Inhibitor of Matrix Metalloprotease -1 (TIMP-1), one of the four natural inhibitors of matrix metalloproteinases was initially identified for its Erythroid Potentiating Activity. Since then, it has been shown to independently induce cell proliferation and inhibit apoptosis in a number of cell types. TIMP-1 has also been shown to promote, but also more often to inhibit angiogenesis. Previously, we have shown that TIMP-1 overexpression in a lung adenocarcinoma cell line (H2009) resulted in more aggressive and vascular tumors in nude mice and increased capillary network formation and tumorigenicity in vitro. An angiogenesis pathway-specific gene array had identified a 3-fold reduction in thrombospondin 1 (TSP-1) levels in the TIMP-1 overexpressing clone HB-1. TSP-1 siRNA clone exhibited a similar increase in in vitro angiogenesis assays. In the present study, we have sought to identify the signaling pathway involved whereby TIMP-1 inhibits TSP-1. To this end, we have used specific inhibitors to inhibit TIMP1 initiated signaling pathways. MAP kinase, PI3/Akt inhibitors were used, either singly or in combination with Focal Adhesion kinase (FAK) inhibitor. Our results indicate a synergistic interaction between PI3/Akt pathway and FAK signaling, which restores TSP-1 levels by approximately 3-fold relative to control. Gelatin zymography of serum-free conditioned media (SFCM) revealed a reduction in proMMP-9 level in the HB-1 clone versus H2009. Additionally MMP9 specific ELISA reveals a two-fold reduction in MMP9 levels. This is in tune with the role of TSP-1 as an inhibitor of MMP-9 activation such that decreased TSP-1 level results in increased activation of MMP-9 with a concomitant reduction in the extracellular level of proMMP-9. Thus our findings define interaction of TIMP-1 via PI3kinase and FAK signaling. resulting in TSP-1 decrease and subsequent downstream promotion of angiogenesis with increased tumor kinetics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5294. doi:1538-7445.AM2012-5294