Temporospatial expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in mouse antigen-induced arthritis

Several lines of evidence speak for an important role of matrix metalloproteinases (MMPs) in the development of progressive joint destruction. To better understand the role of MMPs and their tissue inhibitors (TIMPs) in this process, we have used the antigen-induced arthritis model to study the temporospatial expression of several MMPs and TIMPs during the progression of arthritis. Arthritis was induced by a single intra-articular injection of methylated bovine serum albumin (mBSA) into one or both knee joints of adult mice previously immunised against mBSA. Samples were collected at 3, 7, 21 and 42 days after induction of arthritis for histology and RNA extraction, and analysed by Northern hybridisation, histochemistry and immunohistochemistry for production of several MMPs and TIMPs −1, −2 and −3. A systematic analysis of MMP and TIMP mRNA levels in mouse knee joints demonstrated a general upregulation of both MMPs and TIMPs during progression of arthritis. Upregulation of MMP-9, −13 and −14 coincided with the advancement of cartilage degeneration, but the expression patterns of MMP-9 and −13 also followed the course of synovial inflammation. TIMPs were steadily upregulated throughout the examination period. Immunohistochemical localisation of MMPs and TIMPs suggested the synovium to be the major source of MMP and TIMP production in arthritis, although articular cartilage chondrocytes also showed an increased production of both MMPs and TIMPs.     

Differential expression of transforming growth factor-beta1, connective tissue growth factor,phosphorylated-SMAD2/3 and phosphorylated-ERK1/2 during mouse tooth development

Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta 1 (TGF-β₁) and TGF-β₁-induced CTGF expression is regulated through SMAD and mitogen-activated protein kinase (MAPK) signaling pathways. The fine modulation of TGF-β₁ signaling is very important to the process of tooth development. However, little is known about the localization of CTGF, MAPK and SMAD in the context of TGF-β₁ signaling during odontogenesis. Hence, we aimed to investigate the expression of TGF-β₁, CTGF, phosphorylated-SMAD2/3 (p-SMAD2/3) and phosphorylated-ERK1/2 (p-ERK1/2). ICR mice heads of embryonic (E) day 13.5, E14.5, E16.5, postnatal (PN) day 0.5 and PN3.5 were processed for immunohistochemistry. Results revealed that at E13.5, TGF-β₁ and CTGF were strongly expressed in dental epithelium (DE) and dental mesenchyme (DM), while p-SMAD2/3 was intensely expressed in the internal side of DE. p-ERK1/2 was not present in DE or DM. At E14.5 and E16.5, strong staining for TGF-β₁ and CTGF was detected in enamel knot (EK) and dental papilla (DPL). DPL was intensely stained for p-ERK1/2 but negatively stained for p-SMAD2/3. There was no staining for p-SMAD2/3 and p-ERK1/2 in EK. At PN0.5 and PN3.5, moderate to intense staining for TGF-β₁ and CTGF was evident in preameloblasts (PA), secretary ameloblasts (SA) and dental pulp (DP). p-SMAD2/3 was strongly expressed in SA and DP but sparsely localized in PA. p-ERK1/2 was intensely expressed in DP, although negative staining was observed in PA and SA. These data demonstrate that TGF-β₁ and CTGF show an identical expression pattern, while p-SMAD2/3 and p-ERK1/2 exhibit differential expression, and indicate that p-SMAD2/3 and p-ERK1/2 might play a regulatory role in TGF-β₁ induced CTGF expression during tooth development.     

Dkk1, -2, and -3 expression in mouse craniofacial development

Summary The Dickkopf family is important for embryogenesis and postnatal development and growth. Dkk1 is a strong head inducer and knockout of this gene leads to absence of anterior head structures, which are predominantly formed through neural crest migration. During early craniofacial development, Dkk1 to Dkk3 show developmentally regulated expression in a number of elements. However, their expression and roles in late times of craniofacial development are largely unknown. This study focuses on the expression profile of Dkk1-3 on late embryonic and early postnatal stages. It was found that Dkks were involved in a variety of craniofacial developmental processes, including facial outgrowth, myogenesis, osteogenesis, palatogenesis, olfactory epithelium and tooth development; and the expression persisted to postnatal stage in the muscles and bones. Their expression patterns suggest important roles in these processes; further study is warranted to elucidate these roles.     

Interaction of keratinocytes and fibroblasts modulates the expression of matrix metalloproteinases-2 and -9 and their inhibitors

Disruption of epidermal-mesenchymal communication due to a delay in epithelialization, increases the frequency of developing fibrotic conditions in skin. As matrix metalloproteinases-2 (MMP-2) and -9 (MMP-9) are two key enzymes involved in wound healing and tissue remodeling, here we examined the efficacy of keratinocyte-fibroblast interaction on modulation of these enzymes and their inhibitors. The conditioned media derived from keratinocytes and fibroblasts grown in upper and lower chambers of a co-culture system, respectively, were analyzed for MMP-2 and -9. Keratinocyte or fibroblast conditioned medium (FCM) was used as a control. Gelatinolytic activity analyzed by zymography showed that keratinocytes mainly express MMP-9 and to a lesser extent MMP-2; while fibroblasts express only MMP-2. In a co-culture system, the activities of both MMP-2 and MMP-9 markedly increased in conditioned media collected from bottom chambers. These findings were consistent with the level of MMP-2 and MMP-9 measured by Western blot. Using the same experimental setting, the levels of tissue inhibitors of MMPs (TIMPs) secreted by keratinocytes and fibroblasts grown in the same co-culture system were also evaluated. Western blot showed that fibroblasts secrete only TIMP-1 and TIMP-2 whose levels were increased by co-culturing fibroblasts with keratinocytes. In contrary the level of TIMP-3, which was mainly expressed by keratinocytes, increased by co-culturing these cells with fibroblasts. In conclusion, interaction of fibroblast-keratinocyte modulates the levels of MMP-2 and -9 and their inhibitors produced by these cells and this interaction may be critical for a better healing quality at a late stage of the wound healing process. (Mol Cell Biochem 269: 209–216, 2005)     

Expression of matrix metalloproteinase -2, -9 and tissue inhibitors of metalloproteinase -1, -2, -3 mRNAs in rat uterus during early pregnancy

Zymography and in situ hybridizition were used to investigate matrix metalloproteinase-2, -9 (MMP-2, -9) activities, and expression of mRNAs for MMP-2, -9 and tissue inhibitors of matrix metalloproteinases (TIMP-1, -2, -3) in the rat uterus during early pregnancy (day 1-7). The zymography results showed two forms of MMP-2 (64 and 67 kDa) in the rat uteri during early pregnancy. The 64-kDa MMP-2 activity was the highest on day 2 (P < 0.01) and higher on day 5 and 6 (P < 0.05). The 67-kDa MMP-2 activity reached the highest on day 5 and 6 (P < 0.01). The 64-kDa MMP-2 activity at the implantation sites was higher than those at interimplantation sites (P < 0.05). Furthermore, the 67 kDa MMP-2 can be converted to 64 kDa forms by incubation with p-aminophenylmercuric acetate (APMA) and trypsin in vitro. The 92-kDa MMP-9 activity was only detected on day 5 and 6 of pregnancy (P < 0.01). In situ hybridization showed that on day 1-4 of pregnancy, both MMP-2 and TIMP-2 mRNAs were evidently localized in the basal stromal cells. On day 5, MMP-2 mRNA signals were decreased in the basal stromal cells and mRNA for TIMP-2 was expressed in the epithelial cells and subepithelial stromal cells. The mRNAs for MMP-9, TIMP-1, and -3 were mainly expressed in epithelial cells on day 1-5. At the implantation site on day 6, the mRNAs for MMP-2, -9, TIMP-1, -2, and -3 were highly expressed in the primary decidual zone surrounding the implanting embryo, and in the whole decidualized stromal cells (the primary and secondary decidual zones) at the implantation site on day 7. The intensities of mRNAs for the TIMPs in decidualized stromal cells at the implantation site on day 6 and 7 were stronger than those for the MMPs. The weak mRNAs for MMP-2, -9, TIMP-1, and -3 but not TIMP-2 were also observed in the ectoplacental cone/trophoblastic cells of the implanting embryos. However, at the interimplantation sites on day 6 and 7, MMP-2, -9, TIMP-1, -2, and -3 mRNAs were weakly expressed in the epithelial cells, subepithelial stromal cells, and myometrium. The results suggested that the implanting rat embryo strongly induced MMP-2 and -9 proteins and gene expression for decidulization and embryo invasion, which were strictly controlled and balanced by the simultaneous expression of TIMP-1, -2 and -3.     

Interaction of keratinocytes and fibroblasts modulates the expression of matrix metalloproteinases-2 and -9 and their inhibitors

Disruption of epidermal-mesenchymal communication due to a delay in epithelialization, increases the frequency of developing fibrotic conditions in skin. As matrix metalloproteinases-2 (MMP-2) and -9 (MMP-9) are two key enzymes involved in wound healing and tissue remodeling, here we examined the efficacy of keratinocyte-fibroblast interaction on modulation of these enzymes and their inhibitors. The conditioned media derived from keratinocytes and fibroblasts grown in upper and lower chambers of a co-culture system, respectively, were analyzed for MMP-2 and -9. Keratinocyte or fibroblast conditioned medium (FCM) was used as a control. Gelatinolytic activity analyzed by zymography showed that keratinocytes mainly express MMP-9 and to a lesser extent MMP-2; while fibroblasts express only MMP-2. In a co-culture system, the activities of both MMP-2 and MMP-9 markedly increased in conditioned media collected from bottom chambers. These findings were consistent with the level of MMP-2 and MMP-9 measured by Western blot. Using the same experimental setting, the levels of tissue inhibitors of MMPs (TIMPs) secreted by keratinocytes and fibroblasts grown in the same co-culture system were also evaluated. Western blot showed that fibroblasts secrete only TIMP-1 and TIMP-2 whose levels were increased by co-culturing fibroblasts with keratinocytes. In contrary the level of TIMP-3, which was mainly expressed by keratinocytes, increased by co-culturing these cells with fibroblasts. In conclusion, interaction of fibroblast-keratinocyte modulates the levels of MMP-2 and -9 and their inhibitors produced by these cells and this interaction may be critical for a better healing quality at a late stage of the wound healing process.     

Reference values of concentrations of matrix metalloproteinases-1, -2, -9 and the tissue inhibitor of matrix metalloproteinases (TIMP-1) in the amniotic fluid during physiological pregnancy and delivery

The aim of this study was to determine reference values of matrix metalloproteinases (MMPs) MMP-1, MMP-2, MMP-9 and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in the amniotic fluid at the first stage of labor in physiological pregnancy. Eighty nine women at the first stage of term labor have been examined. Samples of amniotic fluid were taken at the first period of labor by vaginal amniotomy. Concentrations of MMP-1, MMP-2, MMP-9, and TIMP-1 have been investigated in amniotic fluid samples by ELISA kits. The following normal concentration ranges for MMP-1, MMP-2, MMP-9, TIMP-1, and ratios of concentrations of MMPs and TIMP-1 (MMP-1/TIMP-1, MMP-2/TIMP-1, MMP-9/TIMP-1) have been determined for amniotic fluid samples obtained during the first period of labor in physiological pregnancy. These included: MMP-1: 5.1–16.8 pg/mg of protein; MMP-2: 238.3–374.1 pg/mg of protein; MMP-9: 66.1–113.3 pg/mg of protein, TIMP-1: 4.7–13.6 pg/mg of protein, MMP-1/TIMP-1 ratio: 0.1–2.2, MMP-2/TIMP-1 ratio: 19.9–55.7; MMP-9/TIMP-1 ratio: 4.2–17.2.     

Quantitative analysis of matrix metalloproteinases-2 and -9, and their tissue inhibitors-1 and -2 in human placenta throughout gestation

To elucidate the implication of type IV collagenases(MMP-2 and MMP-9) and their tissue inhibitors (TIMP-1 and TIMP-2) for placental development, we quantified their levels in the conditioned media of placental organ culture and primary culture of the trophoblast as well as in the tissue extracts of placentas from different stages of gestation using specific enzyme-linked immunosorbent assays. First trimester villous tissue secreted about 10 times more pro-MMP-2 than pro-MMP-9, and pro-MMP-2 levels dramatically decreased in the second trimester. On the other hand, pro-MMP-9 levels were more than 10 times higher than those of pro-MMP-2 in the primary culture of the first trimester trophoblast, indicating the involvement of stromal cells for prominent pro-MMP-2 secretion from first trimester villous tissue described above. Levels of TIMPs, especially those of TIMP-2, remained constant throughout gestation both in the culture media and tissue extracts. Gelatin zymography revealed abundant secretion of the active form of MMP-2 as well as pro-MMP-2 from first trimester villous tissue. Western immunoblot analysis confirmed the presence of both TIMP-1 and TIMP-2 in placental tissue. These results suggest that active secretion of MMP-2 from villous tissue in the first trimester and constant production of TIMPs throughout gestation are characteristic of placental development.     

Dynamics of activities of matrix metalloproteinases-9 and -2, and the tissue inhibitors of MMPs in fetal fluid compartments during gestation and at parturition in the mare

During late gestation in the mare, rapid fetal growth is accompanied by considerable placental growth and further invasion of the endometrium by microvilli. This growth requires extensive remodeling of the extracellular matrix (ECM). In early pregnancy, we know that matrix metalloproteinase (MMP)-9 and -2 are involved in the endometrial invasion during endometrial cup formation. The present study investigated whether MMPs are found in fetal fluids later in gestation and during parturition, and if there was a difference in their activities between normal and preterm delivery. Amniotic fluids were collected from pony mares during the latter half of gestation, and amniotic and allantoic fluids from pony and thoroughbred mares at foaling. The fluids were analysed for the activity of MMP-9 and -2, and TIMPs using zymography techniques. There was an increase (P = 0.002) in activity of latent MMP-9 when approaching normal foaling, and a decrease (P < 0.001) during foaling. MMP-2 activity did not change through gestation, or during foaling. When comparing samples from pregnancies resulting in preterm deliveries with samples from foaling mares, the activity of MMP-9 was lower (P < 0.001) and MMP-2 activity was higher (P = 0.004) during foaling than preceding preterm delivery. The activity of MMP-9 was lower (P = 0.002) prior to preterm delivery than before delivery of a live foal at term, whereas no difference (P = 0.07) was demonstrated for latent MMP-2 activity when comparing the same groups. The activity of TIMP-2 was higher (P < 0.001) in the pre-parturient period before normal foaling than preceding preterm delivery. These results suggest that MMPs may have a role as markers for high risk pregnancy in the mare.     

Association of metalloproteinases,tissue inhibitors of matrix metalloproteinases,and proteoglycans with development,aging, and osteoarthritis processes in mouse temporomandibular joint

The temporomandibular joint (TMJ) is an important growth and articulation center in the craniofacial complex. In aging it develops spontaneous degenerative osteoarthritic (OA) lesions. Metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPS) play key roles in extracellular matrix remodeling and degradation. Gelatinase activities and immunohistochemical localization of MMP-2, -3, -8, -9, and -13 and TIMP-1 and -2 were examined in mandibular condyle cartilage of neonatal mice up to 18 months old. The most intense immunostaining for all enzymes and TIMPs and the peak of gelatinase activities were found in animals in the stages of early growth (1 week to 3 months) followed by a decrease during maturation and aging. However, clusters of positively immunoreactive chondrocytes were detected in cartilages of old animals displaying OA lesions. Positive safranin-O staining, indicative of sulfated proteoglycans (PGs), was prominent in the TMJ of newborn mice up to 3 months old followed by reduction during maturation and aging, except in regions displaying OA lesions. Temporal codistribution of PGs, MMPs, and TIMPs during skeletal maturation reflected an active growth phase, whereas their reduction coincided with the more quiescent articulating and maintenance phase in the joint cartilage. Osteoarthritic lesions were associated with both increased PG synthesis and MMP immunoreactivity, indicating limited repair activity during initial stages of osteoarthritis.     

Tissue inhibitor of metalloproteinases-2 is expressed in the interstitial matrix in adult mouse organs and during embryonic development.

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a member of a family of inhibitors of matrix-degrading metalloproteinases. A better insight into the role of this inhibitor during development and in organ function was obtained by examining the temporospatial expression of TIMP-2 in mice. Northern blot analysis indicated high levels of TIMP-2 mRNA in the lung, skin, reproductive organs, and brain. Lower levels of expression were found in all other organs with the exception of the liver and gastrointestinal tissue, which were negative of these tissues with complete absence of TIMP-2 mRNA in the epithelium. In the testis, TIMP-2 was present in the Leydig cells, and in the brain, it was expressed in pia matter and in neuronal tissues. TIMP-2 expression in the placenta increased during late gestation and was particularly abundant in spongiotrophoblasts In mouse embryo (day 10.5-18.5), TIMP-2 mRNA was abundant in mesenchymal tissues that surrounded developing epithelia and maturing skeleton. The pattern of expression significantly differs from that observed with TIMP-1 and TIMP-3, therefore, suggesting specific roles for each inhibitor during tissue remodeling and development.     

Prostaglandin E2-dependent enhancement of tissue inhibitors of metalloproteinases-1 production limits dendritic cell migration through extracellular matrix

Dendritic cell (DC) migration is crucial for the initiation of immune responses. The balance between metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) has been shown to modulate DC migration. PGE2, which is overproduced in a wide variety of human malignancies, has been implicated in MMP and TIMP regulation in various cells, including monocytes. In the present study, we hypothesized that tumor-derived PGE2 would affect DC migratory capacity through the extracellular matrix (ECM) by altering MMP and TIMP balance. Treatment of monocyte-derived immature DC with exogenous PGE2 induced TIMP-1 secretion but not MMP-9 production and was correlated with reduced DC migration through ECM. Because recombinant TIMP-1 replicated PGE2 inhibition of DC migration while anti-TIMP-1 neutralizing Ab reversed it, we conclude that PGE2-mediated induction of TIMP-1 was responsible for the reduced migration of PGE2-treated DC. Similarly, DC cultured for 48 h in supernatants from cyclooxygenase-2 overexpressing lung cancer cells that secrete high levels of PGE2, exhibited decreased migration through ECM. Finally, analysis of E prostanoid receptor expression and their selective inhibition revealed that the enhanced TIMP-1 secretion in PGE2-treated DC was mediated predominantly by the E prostanoid receptor 2. These findings indicate that PGE2-dependent enhancement of TIMP-1 production causes reduced migration of DC through ECM.     

Differential regulation of TIMP-1, -2, and -3 mRNA and protein expressions during mouse incisor development

Tissue inhibitors of metalloproteinases (TIMPs) possess multiple functions, in addition to their matrix metalloproteinase (MMP) inhibitory activity. The continuously growing incisor of mouse possesses a stem cell compartment at the apical end of the epithelium (the apical loop) and thus provides an excellent tool to analyze the mechanisms of organogenesis and cytodifferentiation. To understand the functions of TIMPs in tooth development, we have analyzed the gene expression and protein localization of TIMP-1, -2, and -3 during mouse incisor development, from embryonic day 13 (E13) to postnatal day 3 (P3). TIMP-1 was present on the basement membrane during early developmental stages. At P2, TIMP-1 was strongly detected along the apical loop, transiently disappeared from the basement membrane in the cytodifferentiation zone, and later reappeared at the distal end of functional ameloblasts. Expression of TIMP-2 protein was restricted to the outer part of the apical loop throughout the examined stages. At P2, TIMP-2 was present on the basement membrane at the outer part of the apical loop. The dental follicle also expressed Timp-2, and the corresponding protein was abundant within the extracellular matrix. Timp-3 mRNA was highly expressed in the mesenchyme surrounding the apical loop. During matrix formation, Timp-3 was expressed by subodontoblasts, and the protein was detected in this layer and between odontoblasts. Distinct temporal and spatial expression patterns of TIMPs suggest divergent functions of these factors in incisor organogenesis. This work was supported by INSERM, CNRS, ARC, French Ministry of Research (ACI), Japanese Ministry of Education, Culture, Sports, Science, and Technology, and Niigata University Research Projects.     

Effects of matrix proteins on the expression of matrix metalloproteinase-2, -9, and -14 and tissue inhibitors of metalloproteinases in human cytotrophoblast cells during the first trimester.

The activity of matrix metalloproteinases (MMPs) specifies the ability of the trophoblast cell to degrade extracellular matrix (ECM) substrates. Usually the process of normal human placentation involves a coordinated interaction between the fetal-derived trophoblast cells and their microenvironment in the uterus. In this study, the effects of ECM proteins on the expression of MMP-2, -9, and -14 (membrane-type MMP-1); and the production of tissue inhibitors of metalloproteinase (TIMP) types -1, -2, and -3 have been investigated. Cytotrophoblast cells at 9 or 10 wk of gestation were cultured on various ECM coated dishes under serum-free conditions. Gelatin zymography analysis showed that cells grown on fibronectin (FN), laminin (LN), and vitronectin (VN) secreted more MMP-9 (about 1.5- to 3-fold more) than cells cultured on collagen I (Col I), whereas the secretion of MMP-9 by cells cultured on collagen IV (Col IV) was only half that by the cells on Col I. Northern Blot analysis gave the same results as zymography, indicating that expression of the MMP-9 gene in cytotrophoblast cells can be affected by matrix proteins. There was no significant difference in the expression of MMP-2 either at protein or mRNA levels among the cells cultured on the different matrix substrates. The expression of MMP-14 was regulated in a manner similar to that of MMP-2. Using ELISA, we detected higher levels of TIMP-1 in the culture medium of cells grown on VN, LN, and FN compared with that grown on Col I. But the expression of TIMP-3 mRNA was remarkably inhibited by VN, and ECM proteins had no effect on TIMP-1 and TIMP-2 mRNA expression. It was also observed that cultured cytotrophoblast cells expressed the corresponding receptors for the tested matrix proteins, such as integrins alpha(1), alpha(5), alpha(6), beta(1), and beta(4). Furthermore, the adhesiveness of cytotrophoblast cells on Col I, Col IV, FN, and LN was increased by 62%, 45%, 21%, and 22%, respectively, when compared with adhesiveness on VN. Isolated cytotrophoblast cells remained stationary when cultured on dishes coated with Col I and Col IV, but they assumed a more motile morphology and aggregated into a network when cultured on LN and VN. These data indicate that human trophoblast cells interact with their microenvironment to control their behavior and function.     

Regional changes in CNS levels of the S-100 and 14-3-2 proteins during development and aging of the mouse

The levels of the S-100 and 14-3-2 proteins were determined in a number of regions of mouse brain at intervals from 1 day to 30 months of age. Both S-100 and 14-3-2 were found in measurable amounts as early as the first day of postnatal age but did not begin to accumulate rapidly in the forebrain, brain stem and cerebellum of the mouse brain until some time between the 7th and 14th days. From days 14 to 28 the levels of S-100 and 14-3-2 in each region continued to increase rapidly with the exception of the forebrain where the rate of accumulation of S-100 appeared to lag considerably behind that in the other regions. The proteins continued to accumulate at a rapid rate until approximately 6 months of age. From 6 to 30 months of age, the levels of 14-3-2 remained relatively stable in cerebellum, hippocampus and hypothalamus and appeared to decrease slightly in striatum and cerebral cortex. In the case of S-100, the level of the protein increased in all regions of brain from 6 to 30 months but the increase was most pronounced in the hippocampus, hypothalamus and striatum. The principal conclusion derived from this study is that the biochemical development and aging of the central nervous system are regionally selective processes.