Selective progesterone receptor modulator asoprisnil down-regulates collagen synthesis in cultured human uterine leiomyoma cells through up-regulating extracellular matrix metalloproteinase inducer

BACKGROUND: A recent clinical trial demonstrated that selective progesterone receptor modulator asoprisnil is effective in reducing uterine leiomyoma volume. We investigated the effects of asoprisnil in vitro on the expression of the extracellular matrix (ECM)-remodeling enzymes and collagens in cultured leiomyoma and matching normal myometrial cells. METHODS: The expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagens were assessed by western blot analysis. RESULTS: Untreated cultured leiomyoma cells had significantly lower EMMPRIN (P < 0.05), MMP-1 (P < 0.05) and membrane type 1-MMP (MT1-MMP) (P < 0.01) protein contents, but significantly higher TIMP-1 (P < 0.05), TIMP-2 (P < 0.01), type I (P < 0.05) and type III (P < 0.01) collagen protein contents compared with untreated cultured myometrial cells. Treatment with asoprisnil at concentrations > or =10(-7) M for 48 h significantly (P < 0.05) increased EMMPRIN, MMP-1 and MT1-MMP protein contents, and decreased TIMP-1 (P < 0.05), TIMP-2 (P < 0.01), type I (P < 0.01) and type III (P < 0.05 at 10(-7) M; P < 0.01 at 10(-6) M) collagen protein contents in cultured leiomyoma cells compared with control cultures. However, asoprisnil treatment did not affect the protein contents of ECM-remodeling enzymes and collagens in cultured myometrial cells. CONCLUSIONS: These results suggest that asoprisnil may reduce collagen deposit in the ECM of cultured leiomyoma cells through decreasing collagen synthesis and enhancing the expression of EMMPRIN, MMPs and TIMPs without comparable effects on cultured myometrial cells.     

Progesterone receptor modulator CDB-2914 induces extracellular matrix metalloproteinase inducer in cultured human uterine leiomyoma cells

Effects of progesterone receptor modulator CDB-2914 on the expression of the extracellular matrix (ECM) components were examined in cultured human uterine leiomyoma and myometrial cells. ECM metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagen levels were assessed by Western blot analysis, MMP activity assay and real-time RT-PCR. RNA interference (RNAi) of EMMPRIN was performed using small interfering mRNA. In cultured leiomyoma cells, CDB-2914 treatment at concentrations greater than or equal to 10(-8) M significantly increased EMMPRIN, MMP-1 and MMP-8 protein contents and MMP-1, MMP-2, MMP-3 and MMP-9 mRNA levels, and activity of MMP-1, MMP-2, MMP-3 and MMP-9 in the medium. TIMP-1 and TIMP-2 were significantly decreased at mRNA and protein levels by CDB-2914 treatment at concentrations > or =10(-7) M in these cells. CDB-2914 treatment decreased types I and III collagen protein contents. However, CDB-2914 treatment did not affect the ECM component expression in cultured myometrial cells. RNAi of EMMPRIN abrogated CDB-2914-mediated both induction of MMPs and reduction of TIMPs and collagens in cultured leiomyoma cells. These results suggest that CDB-2914 modulates the expression of EMMPRIN, MMPs, TIMPs and collagens in cultured leiomyoma cells without comparable effects on myometrial cells.     

基质金属蛋白酶及其组织抑制因子在人前列腺组织及各种类型前列腺细胞中的表达

目的：研究基质金属蛋白酶（MMPs）及其组织抑制因子（TIMPs）在人前列腺组织及各种类型细胞中的表达。方法： 用半定量RT－PCR的方法，对癌变和非癌变部分的前列腺组织、原代培养的平滑肌细胞、成纤维细胞、上皮细胞以及4种前列腺上皮细胞系（BPH－1、LNCaP、DU－145和PC－3）中MMP2、MMP7和MMP9、膜型基质金属蛋白酶1和3（MT1－MMP和MT3－MMP）及其组织抑制因子1和2（TIMP－1和TIMP－2）的mRNA 水平进行了测定。结果：MMP-2主要在前列腺基质细胞中表达；MMP-7和MMP-9则在前列腺上皮细胞中有较高的表达；MT1-MMP、MT3-MMP、TIMP-1和TIMP-2在前列腺基质细胞和上皮细胞中均有表达，但MT1-MMP和MT3-MMP在成纤维细胞中的表达量较高；另外，各种基质金属蛋白酶及其组织抑制因子在各种前列腺细胞系中也存在差异表达。结论： MMPs和TIMPs在前列腺组织及其各种类型细胞中的差异表达提示：它们可能在前列腺癌的转移中起着不同的作用。     

Regulation of matrix metalloproteinases and their inhibitors in the left ventricular myocardium of patients with aortic stenosis

Aortic stenosis (AS) results in myocyte and extracellular matrix remodeling in the human left ventricle (LV). The myocardial renin-angiotensin system is activated and collagens I and III and fibronectin accumulate. We determined the yet unknown regulation of enzymes that control collagen turnover, i.e., LV matrix metalloproteinases (MMP) and their tissue inhibitors (TIMPs) in human AS. We compared LV samples from AS patients undergoing elective aortic valve replacement (n=19) with nonused donor hearts with normal LV function (controls, n=12). MMP-2, MMP-9, MT1-MMP, and extracellular matrix metalloproteinase inducer (EMMPRIN), TIMP-1, TIMP-2, TIMP-3, and TIMP-4 mRNA were quantitated by real-time RCR. MMP-1, MMP-2, MMP-3, TIMP-3, TIMP-4, and EMMPRIN protein were measured by immunoblotting and MMP-9 and TIMP-1 protein by ELISA. Gelatinolytic MMP-2 and MMP-9 activity was measured by zymography. MMP-2 was increased in AS at mRNA, protein, and activity levels (131%, 193%, and 138% of controls). MMP-3 protein (308%) and EMMPRIN mRNA and protein were also upregulated (171% and 200%). In contrast, MMP-1 (37%) and MMP-9 mRNA, protein, and activity (26%, 21%, and 52%) were downregulated. MMP-9 activity was inversely correlated with LV size. TIMP-1 mRNA and protein were decreased (55% and 73%). In contrast, TIMP-2 mRNA (358%), TIMP-3 mRNA and protein (145% and 249%) were increased. TIMP-4 mRNA was not altered, but TIMP-4 protein was upregulated to 350%. Changes were similar in AS patients with normal and impaired LV ejection fraction. The dysregulation of myocardial MMPs and TIMPs in human AS starts at an early disease stage when LV function is still normal. In spite of upregulation of some MMPs the balance between MMP and TIMP is shifted towards MMP inhibition in human AS and may contribute to collagen accumulation.     

Identification of a calcium-dependent matrix metalloproteinase complex in rat chorioallantoid membranes during labour

The induction of the expression of matrix metalloproteinases (MMPs) and their extracellular activation are key processes in connective tissue degradation in the chorioallantoid membrane during rat labour. However, the regulatory mechanisms remain largely unknown. Here, we report the identification of a calcium-dependent high molecular weight complex composed of MMP-9, MMP-3, MMP-2, tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2, identified by zymography and western blotting. Molecular sieve chromatography confirmed the presence of a complex of MMPs and TIMPs with an exclusion volume >670 kDa. Differential scanning calorimetry of the complex confirmed the existence of a macromolecular complex that unfolds with a broad transition; it is denatured over a wide range of temperatures and has a T(m) of 72 degrees C in the presence of Ca(2+). When denatured in the absence of Ca(2+), there were at least eight transitions with T(m)s that corresponded to pro-MMP-9, MMP-9, pro-MMP-3, MMP-3, pro-MMP-2, MMP-2, TIMP-1 and TIMP-2. Co-localization of the same molecular components was demonstrated by confocal microscopy using cell-depleted chorioallantoid membranes. The assembly and disassembly of the complex can be reproduced at physiological concentrations of Ca(2+). This complex provides a potential mechanism for the enzymatic regulation of MMPs, which may participate in connective tissue degradation leading to the rupture of the fetal membranes during labour.     

Immunohistochemical study of metalloproteinases and their tissue inhibitors in the lungs of patients with diffuse alveolar damage and idiopathic pulmonary fibrosis.

Immunohistochemical and confocal microscopic studies of the localization of matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and type IV collagen were made in lung tissues from patients with normal pulmonary histology (n = 3), diffuse alveolar damage (n = 14), and idiopathic pulmonary fibrosis (n = 12). Pretreatment with pepsin revealed otherwise undetectable MMP- and TIMP-immunoreactive sites. In normal lung, MMP-2, MMP-9, TIMP-1, and TIMP-2 were localized in ciliated cells, endothelial cells, pneumocytes, macrophages, and smooth muscle cells; fibroblasts showed a strong reaction only for MMP-2. Only TIMP-2 showed co-localization with type IV collagen. Myofibroblasts and epithelial cells expressed increased reactivity for MMPs and TIMPs in both disorders. The reactivities for MMPs and TIMPs were stronger in diffuse alveolar damage. MMP-2 showed focal co-localization in capillary endothelial and disrupted epithelial basement membranes, suggesting activation of collagenolysis. A protective effect against this lysis was suggested by the extensive co-localization of TIMP-2 with type IV collagen and fibrillar collagens. Alveolar buds showed increased reactivity for MMPs and TIMPs in their lining epithelial cells, myofibroblasts, and their basement membranes; however, their matrices were mostly unreactive. These findings emphasize the complexity of the roles of MMPs and TIMPs in collagen turnover in diffuse alvcolar damage and idiopathic pulmonary fibrosis.     

Gelatin zymography and substrate cleavage assays of matrix metalloproteinase-2 in breast carcinoma cells overexpressing membrane type-1 matrix metalloproteinase

Gelatin zymography is the common method for examining matrix metalloproteinase-2 (MMP-2) in cells and media samples. Activation of the latent MMP-2 zymogen involves its binding to the cell surface MT1-MMP*TIMP-2 (membrane type-1 matrix metalloproteinase/tissue inhibitor of matrix metalloproteinase-2) complex with subsequent cleavage of proMMP-2 by TIMP-2-free adjacent MT1-MMP. This is followed by autolytic maturation of the activation intermediate and the release of the mature MMP-2 species from cell surfaces into the extracellular milieu. To observe the MMP-2 activation pathway in more detail, proMMP-2-deficient MCF7 breast carcinoma cells expressing MT1-MMP were incubated with excess proMMP-2 to saturate the available MT1-MMP*TIMP-2 surface receptors. After removal of the unbound material, the kinetics of proMMP-2 activation and MMP-2 release from cells into media was monitored by gelatin zymography and substrate cleavage. Our observations demonstrate that gelatin zymography is insufficient for providing meaningful information about the status of MMP-2. The proteolytically competent mature MMP-2 moiety alone, but not in its complex with TIMP-2, was released from the cells. In tissue culture conditions, the enzyme's proteolytic activity was suppressed in the next 30 to 60 minutes by tissue inhibitors of MMPs, especially by TIMP-1. The picture emerges that there is a likely temporal regulation of MMP-2 activity by TIMPs in tumor cells. These relatively rapid changes of the MMP-2 status cannot be detected by gelatin zymography. Additional studies are needed to examine the significance of this phenomenon in vivo.     

Dynamic expression of mRNAs and proteins for matrix metalloproteinases and their tissue inhibitors in the primate corpus luteum during the menstrual cycle

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) may be involved in tissue remodelling in the primate corpus luteum (CL). MMP/TIMP mRNA and protein patterns were examined using real-time PCR and immunohistochemistry in the early, mid-, mid-late, late and very late CL of rhesus monkeys. MMP-1 (interstitial collagenase) mRNA expression peaked (by >7-fold) in the early CL. MMP-9 (gelatinase B) mRNA expression was low in the early CL, but increased 41-fold by the very late stage. MMP-2 (gelatinase A) mRNA expression tended to increase in late CL. TIMP-1 mRNA was highly expressed in the CL, until declining 21-fold by the very late stage. TIMP-2 mRNA expression was high through the mid-luteal phase. MMP-1 protein was detected by immunocytochemistry in early steroidogenic cells. MMP-2 protein was prominent in late, but not early CL microvasculature. MMP-9 protein was noted in early CL and labelling increased in later stage steroidogenic cells. TIMP-1 and -2 proteins were detected in steroidogenic cells at all stages. Thus, MMPs and TIMPs are dynamically expressed in a cell-specific manner in the primate CL. Early expression of MMP-1 is suggestive of a role in tissue remodelling associated with luteinization, whereas MMP-2 and -9 may contribute to later stage luteolysis. TIMP expression may control MMP activity, until declining at luteolysis.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in joint fluid of the patients with loose artificial hip joints.

The pseudojoint cavity formed in patients undergoing total hip arthroplasty (THA) is later remodeled to synovial membrane-like tissue, which produces pseudosynovial fluid. This pseudosynovium also is an important source of matrix metalloproteinases (MMPs). As it is widely speculated that synovial fluid MMPs may contribute to local tissue degradation in rheumatoid arthritis (RA) and osteoarthritis (OA), we hypothesize that locally produced MMPs are found in the pseudosynovial fluid, via which they have access to the implant-host interface, and that if they retain their proteolytic potential, they might contribute to aseptic loosening. Enzyme-linked immunosorbent assay (ELISA), immunoblotting, and zymography were used to analyze MMPs and tissue inhibitors of metalloproteinases (TIMPs) in synovial fluid in aseptic loosening, which was compared to RA and OA. Pseudosynovial THA fluid was characterized using low levels of MMP-1 but moderate levels of MMP-13 and MT1-MMP (MMP-14). Due to the lack of an appropriate assay, MMP-13 and MT1-MMP were not similarly assessed, but the immunoblotting indicated that they were in the 56 kD intermediate proteolytically processed forms. The MMP-9 level was intermediate between RA and OA. MMP-2 was on a significant level, but there were no differences among study groups. The THA group also was characterized using relatively high levels of TIMP-1 and TIMP-2. Accordingly, MMP-9 and MMP-2 were found to occur in the 92 kD and 72 kD proenzyme form, respectively, with full activity retained in all study groups. The data suggest that proMMP-2-TIMP-2 and proMMP-9-TIMP-1 complexes are formed in the pseudosynovial fluid due to the excess of TIMPs over MMPs in aseptic loosening of THA. TIMP-complexed MMPs are resistant to MMP-mediated proteolytic activation, which may explain their latency and proenzyme zymogen form. Thus, formation of stabilizing proMMP-TIMP complexes enable transportation of proMMPs far from their original site of production. Due to motion-associated cyclic changes of the intra-articular pressure, fluid-phase MMPs stabilized by TIMPs might be absorbed to implant surfaces and interface tissues and help to dissect the implant/cement-to-bone interface in situ. Consequently, they may contribute to local proteolytic/tissue destructive events and aseptic loosening.     

Protein kinase C activation by phorbol ester increases in vitro invasion through regulation of matrix metalloproteinases/tissue inhibitors of metalloproteinases system in D54 human glioblastoma cells

To elucidate possible mechanisms of phorbol 12-myristate 13-acetate (PMA) induced in vitro invasiveness of glioblastoma cells, we examined expression levels of membrane-type 1 matrix metalloproteinase (MT1-MMP), MMP-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 using Western blotting and gelatin zymography assay, and found that PMA induced the secretion of MMP-9, activated MMP-2 proenzyme to fully active form of 59 kDa, down-regulated the TIMP-1 and TIMP-2 secretion, and increased MT1-MMP on the cell surface. However, PKC inhibitor Go 6983 reversed all of these effects brought about by PMA. We, therefore, conclude the activation of PKC by PMA in these cells plays a critical role in the regulation of MMPs/TIMPs system, which has a major role in tumor invasion and metastasis.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human chondrosarcomas

The aim of the present study was to characterise the ability of malignant chondrosarcomas to invade normal bone by analysing their production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). For this purpose 12 chondrosarcomas were investigated for the expression of mRNAs for several MMPs and all 4 TIMPs by Northern hybridisation, and for immunohistochemical localisation of the proteins. A characteristic finding of these analyses was increased expression of MMP-13, MMP-14 and TIMP-2 mRNAs in chondrosarcomas when compared with nonmalignant control samples. Individual chondrosarcomas also exhibited elevated levels of MMP-1, MMP-7 and MMP-9 mRNAs. The results of Northern hybridisations were supported by immunohistochemical stainings of the corresponding tumour areas for MMP-2, MMP-14 and TIMP-2, further suggesting that these may have prognostic value for determining whether individual chondrosarcomas are locally aggressive or have a probability of recurrence. Another finding of the present study was a marked heterogeneity in histologic appearance and gene expression of the chondrosarcomas, emphasising the importance of analysing several areas of these tumours to get representative results. These findings suggest that analysis of MMPs could be a useful diagnostic indicator in patients with cartilaginous tumours and could help in differentiating between a low-grade malignant chondrosarcoma and a benign growing enchondroma.     

Gelatinases and their tissue inhibitors during human ovulation: increased expression of tissue inhibitor of matrix metalloproteinase-1

Remodelling of the extracellular matrix (ECM) of the follicular wall by matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) has been suggested to be crucial in ovulation. To investigate the expression of the gelatinases, MMP-2 and MMP-9, together with their inhibitors, TIMP-2 and TIMP-1, in the perifollicular ovarian stroma from women just before and during ovulation, we obtained biopsies of the stroma adjacent to the leading follicle. Laparoscopic surgery was performed either before the LH peak or at any of three intervals after ovulation triggering by hCG. Immunoblotting, immunohistochemistry and quantitative RT-PCR were performed. All four proteins were expressed by immunoblots, with no detectable changes in the expression of MMP-2, MMP-9 and TIMP-2. Scattered immunostaining for MMP-9 and TIMP-2 was seen, and MMP-2 was demonstrated in a concentric layer. A significant increase in TIMP-1 protein and mRNA was seen during the three ovulatory phases, and a strong and patchy immunostaining for TIMP-1 was shown. This is the first study that has demonstrated an ovulation-associated expression of these ECM-remodelling enzymes around the human follicle at ovulation. The increased expression of TIMP-1 may reflect a specific temporal inhibition of collagenolysis and thereby a time-dependent regulation of ECM breakdown in areas surrounding the apex of the follicle.     

Metalloproteinases in juvenile angiofibroma--a collagen rich tumor

Matrix metalloproteinases (MMPs) act in diverse physiological and pathological conditions such as tumor growth and angiogenesis by cleaving extracellular matrix and nonmatrix substrates. MMPs with gelatinase/collagenase activity have not yet been studied in juvenile angiofibroma, a unique fibrovascular tumor with prominent collagen expression. Quantitative real-time polymerase chain reaction studies, Western blot analysis, immunofluorescence studies, gel zymography, and in situ zymography were used to analyze MMP-1, MMP-2, MMP-9, MMP-13, MMP-14, TIMP-1, and TIMP-2 in 9 juvenile angiofibromas and 2 inferior nasal turbinate specimens. Quantitative real-time polymerase chain reaction found significantly elevated expression of MMP-2, MMP-9, and MMP-14 (P < .05) in tumor tissue compared with the inferior nasal turbinate specimens. Western blot analysis detected more prominent MMP-1, MMP-2, and MMP-9 protein levels in juvenile angiofibromas compared with inferior nasal turbinates, but not MMP-13, MMP-14, TIMP-1, and TIMP-2. Immunofluorescent staining proved a mainly stromal localization of the analyzed MMPs. Only MMP-9 and MMP-14 were also detected in vessel walls. MMP-1, MMP-2, and MMP-13 also stained mast cells. Gel zymography indicated increased MMP-2 and MMP-9 gelatinase activity in juvenile angiofibromas compared with inferior nasal turbinates. Finally, in situ zymography detected very high stromal gelatinase/collagenase activity. This study indicates significant expression of MMPs with gelatinase/collagenase activity in juvenile angiofibromas with evidence of a disturbed balance of MMPs to TIMPs toward enhanced MMP activity. These MMPs are assumed to be involved in tumor pathology with an influence on tumor growth and angiogenesis.     

Secretion of matrix metalloproteinase-2, matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases into the intrauterine compartments during early pregnancy.

Matrix metalloproteinases (MMPs) are important enzymes in tissue remodelling, a key event for the development of the fetal membranes and placenta and establishing the feto-maternal interface during early pregnancy. This study has examined the secretion of the gelatinases, MMP-2 (72 kDa) and MMP-9 (92 kDa), and the endogenous tissue inhibitors of metalloproteinases (TIMPs) into extra-embryonic coelomic and amniotic fluids, the two principal intra-uterine compartments of the first trimester, and compared them to amniotic fluid collected later in gestation. In extra-embryonic coelomic fluid, gelatin zymography demonstrated that MMP-2 (72 kDa) was the predominant gelatinase, with some MMP-9 present. A broad range of TIMPs corresponding to TIMP-1 and TIMP-2, glycosylated and unglycosylated TIMP-3 and TIMP-4 was detected in this compartment by reverse zymography and immunoblot analyses. There was little gelatinase or TIMP activity in amniotic fluid in the first trimester. In amniotic fluid from the second trimester after fusion of the membranes obliterating the extra-embryonic coelom, and at term elective caesarean section, MMP-2 is the predominant gelatinase present, with a broad spectrum of TIMPs. These findings demonstrate that predominantly MMP-2 and also MMP-9, regulated by a range of TIMPs, are involved in intra-uterine tissue remodelling during the establishment of pregnancy.     

Increased levels of serum tissue inhibitor of metalloproteinase-1 but not metalloproteinase-3 in atopic dermatitis

Matrix metalloproteinases and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs), contribute to inflammation-induced tissue destruction and subsequent remodeling for maintenance of tissue homeostasis. Since the production of these enzymes and their inhibitors is regulated by mediators such as proinflammatory cytokines and growth factors, elevated levels of serum TIMPs and/or MMPs have been documented in patients with several inflammatory disorders. In this study, we examined the role of TIMPs and MMPs in the pathogenesis of atopic dermatitis (AD) by evaluating the serum levels of TIMP-1 and MMP-3 in 40 patients with AD and 20 control subjects by ELISA. The serum TIMP-1 levels were significantly higher in AD patients in exacerbation status than in nonatopic subjects, whereas serum MMP-3 levels were not significantly different between them. As a result, AD patients revealed significantly elevated TIMP-1/MMP-3 ratios. The levels of serum TIMP-1 were significantly reduced in AD patients following conventional treatments. Significantly higher values of peripheral eosinophil counts, serum levels of IgE and lactate dehydrogenase, eruption score, and eruption area were noted in the AD patients with elevated TIMP-1 levels when compared with those with normal values. Moreover, the points of chronic eruptions such as lichenification and prurigo were significantly higher in the patients with elevated TIMP-1 levels than those with normal TIMP-1, while those of acute lesions such as oozy/microvesicles and oedema were not different between these groups. Serum TIMP-1 level may be a useful marker to estimate the long-term disease activity of AD.     

Temporal changes in collagen composition and metabolism during rodent palatogenesis

Cleft lip and palate is a common craniofacial malformation in man. The aetiology is multifactorial and not known. Since collagen is a major structural component of the developing palate, we studied its composition and metabolism during palate shelf formation and elevation in the rat. Palatal shelves were harvested at embryonic days (E) 15, 16 and 17 as well as post-partum. Palatal collagen increased threefold from E15 to E17 and tenfold from E17 to 5-day-old pups. Palatal calcification was seen in the main, post-partum. Collagen cross-linking, which may be important in shelf elevation and union, varied. The concentration of hydroxylysyl-pyridinolone cross-links was greatest prior to shelf elevation, declining thereafter. Similarly, the highest concentration of dihydroxylysinononorleucine was seen at E16 and this supports the concept of a compliant mesenchymal shelf responding to an intrinsic elevating force. We then determined if enzymes responsible for matrix degradation, matrix metalloproteinases (MMP) and the tissue inhibitors of metalloproteinases (TIMPs) altered over the same time periods. MMP-2, and TIMP-1 and TIMP-2 were identified by gelatin zymography and reverse zymography, respectively. MMP-3 activity was determined with a fluorogenic substrate assay. TIMP-1, TIMP-2 and MMP-3 levels remained constant from E15 to E17. The MMP-2 levels showed a significant elevation from E15 to E16 and E16 to E17. This suggests the regulation of extracellular matrix is likely to be of importance in palate morphogenesis.