Diminished matrix metalloproteinase 2 (MMP-2) in ectomesenchyme-derived tissues of the Patch mutant mouse: regulation of MMP-2 by PDGF and effects on mesenchymal cell migration.

Platelet-derived growth factors (PDGF) regulate cell proliferation, survival, morphology, and migration, as well as deposition and turnover of the extracellular matrix. Important roles for the A form of PDGF (PDGF-A) during connective tissue morphogenesis have been highlighted by the murine Patch mutation, which includes a deletion of the alpha subunit of the PDGF receptor. Homozygous (Ph/Ph) embryos exhibit multiple connective tissue defects including cleft face (involving the first branchial arch and frontonasal processes), incomplete heart septation, and heart valve abnormalities before they die in utero. Analyses of the cell biology underlying the defects in Ph/Ph embryos have revealed a deficit in a matrix metalloproteinase (MMP-2) and one of its activators (MT-MMP) that are likely to be involved in cell migration and tissue remodeling, two processes necessary for normal cardiac and craniofacial development. Morphogenesis of these structures requires infiltration of ectomesenchymal precursors and their subsequent deposition and remodeling of extracellular matrix components. First branchial arch and heart tissue from E10.5 embryos were examined by gelatin zymography and RT-PCR in order to characterize the expression of MMPs in these tissues. Of the MMPs examined, only MMP-2 and one of its activators, MT-MMP, were expressed in the first arch and heart at this stage of development. Tissues from Ph/Ph embryos exhibited a significant decrease in both MMP-2 and MT-MMP compared to tissues from normal embryos of the same developmental stage. In order to assess whether this decrease affects the motile activity of mesenchymal cells, cell migration from Ph/Ph branchial arch explants was compared to migration from normal arch tissue and found to be significantly less. In addition, the migratory ability of branchial arch cells from normal explants could be reduced in a similar manner using a specific MMP inhibitor. Although it is still unclear whether the MMP-2 reduction is a direct result of the absence of response of Ph/Ph cells to PDGF-A treatment of normal branchial arch cells in vitro with recombinant PDGF-AA significantly upregulated MMP-2 protein. Together, these results suggest that PDGF-A regulates MMP-2 expression and activation during normal development and that faulty proteinase expression may be at least partially responsible for the developmental defects exhibited by Ph/Ph embryos.     

Expression of matrix metalloproteinase-9 (gelatinase B) in gouty arthritis and stimulation of MMP-9 by urate crystals in macrophages

To investigate the relevance of gelatinase-B (matrix metalloproteinase 9, MMP-9) in gouty arthritis (GA), we tested the occurrence of MMP-9 in GA patients and cell culture system. Gelatinolytic activity in the synovial fluid (SF) of patients with different kinds of arthritis was assessed by gelatin zymography. A predominant 92-kDa MMP-9 gelatinolytic activity was evident in rheumatoid arthritis (RA) and GA samples, but no activity was observed in osteoarthritis (OA) samples. Among the 53 SF samples (9 RA, 24 GA, and 20 OA) analyzed for MMP-9 and tissue inhibitor of metalloproteinase (TIMP-1) antigen levels by ELISA, MMP-9 antigen levels were elevated tenfold in GA SF compared with OA SF. In addition, GA synovial tissue extracts revealed elevated levels of MMP-9 expression as compared to OA tissue extracts by Western blot and RT-PCR analysis. Immunohistochemical studies demonstrated that MMP-9 immunoreactivity was more intense in GA than in OA synovial tissues. Furthermore, macrophages activation by gouty crystals in vitro was examined. Crystals stimulated MMP-9 gene expression in macrophage cell line and such stimulation was suppressed by PD98059. These findings suggest that the abnormal production of MMP-9 by macrophages is a reflection of the pathological conditions in joints of patients with GA, and that the activation of MMP-9 in the joint is known to play an important role in joint disease.     

PDGF-C controls proliferation and is down-regulated by retinoic acid in mouse embryonic palatal mesenchymal cells

BACKGROUND: Platelet-derived growth factor C (PDGF-C) was recently identified as a member of the PDGF ligand family. Some observation suggests that PDGF-C could play an important role in palatogenesis highlighted by the Pdgfc(-/-) mouse with cleft palate, which led us to examine the mechanism of PDGF-C signaling in palatogenesis. It is well known that retinoic acid (RA) is a teratogen that can effectively induce cleft palate in the mouse. Due to the critical roles of PDGF-C and RA in cleft palate, the link between cleft palate induced by RA and loss of PDGF-C was investigated. METHODS: Retarded mesenchymal proliferation is an important cause for cleft palate. To clarify the mechanism of PDGF-C in palatogenesis, we evaluated the effects of PDGF-C and anti-PDGF-C neutralizing antibody on proliferation activity in mouse embryonic palatal mesenchymal (MEPM) cells. RESULTS: Briefly, our results show PDGF-C promotes proliferation, anti-PDGF-C antibody inhibits it in MEPM cells, and RA downregulates the PDGF-C expression both at the mRNA and protein levels. CONCLUSIONS: These demonstrate that PDGF-C is a potent mitogen for MEPM cells, implying that inactivated PDGF-C by gene-targeting or reduced PDGF-C by RA may both cause inhibition of proliferation in palatal shelves, which might account for the pathogenesis of cleft palate in Pdgfc(-/-) mouse or RA-treated mouse. In conclusion, our results suggest that PDGF-C signaling is a new mechanism of cleft palate induced by RA.     

Differential regulation of metalloproteinase production, proliferation and chemotaxis of human lung fibroblasts by PDGF, interleukin-1beta and TNF-alpha

Fibroblast migration, proliferation, extracellular matrix protein synthesis and degradation, all of which play important roles in inflammation, are themselves induced by various growth factors and cytokines. Less is known about the interaction of these substances on lung fibroblast function in pulmonary fibrosis. The goal of this study was to investigate the effects of PDGF alone and in combination with IL-1beta and TNF-alpha on the production of human lung fibroblast matrix metalloproteinases, proliferation, and the chemotactic response. The assay for MMPs activity against FITC labeled type I and IV collagen was based on the specificity of the enzyme cleavage of collagen. Caseinolytis and gelatinolytic activities of secreted proteinases were analyzed by zymography. Fibronectin in conditioned media was measured using human lung fibronectin enzyme immunoassay. Cell proliferation was measured by 3H-Thymidine incorporation assay. Cell culture supernatants were tested for PGE2 content by ELISA. Chemotactic activity was measured using the modified Boyden chamber. Matrix metalloproteinase assay indicated that IL-1beta, TNF-alpha and PDGF induced intestitial collagenase (MMP-1) production. MMP assay also indicated that IL-1beta and TNF-alpha had inhibitory effects on MMP-2,9(gelatinaseA,B) production. Casein zymography confirmed that IL-1beta stimulated stromlysin (matrix metalloproteinase 3; MMP-3) and gelatin zymography demonstrated that TNF-alpha induced MMP-9 production in human lung fibroblast, whereas PDGF alone did not. PDGF in combination with IL-1beta and TNF-alpha induced MMP-3 and MMP-9 activity, as demonstrated by zymography. PDGF stimulated lung fibroblast proliferation in a concentration-dependent manner, whereas IL-1beta and TNF-alpha alone had no effect. In contrast, the proliferation of human lung fibroblasts by PDGF was inhibited in the presence of IL-1beta and TNF-alpha, and this inhibition was not a consequence of any elevation of PGE2. PDGF stimulated fibroblast chemotaxis in a concentration-dependent manner, and this stimulation was augmented by combining PDGF with IL-1beta and TNF-alpha. These findings suggested that PDGF differentially regulated MMPs production in combination with cytokines, and further that MMP assay and zymography had differential sensitivity for detecting MMPs. The presence of cytokines with PDGF appears to modulate the proliferation and chemotaxis of human lung fibroblasts.     

Fusion expression of DDR2 extracellular domain in insect cells and its purification and function characterization

Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, a ligand for DDR2, up-regulates matrix metalloproteinase 1 (MMP-1) and MMP-2 expression in extracellular matrix (ECM). To investigate the role of DDR2 in cartilage destruction in rheumatoid arthritis (RA), we expressed the extracellular domain (ECD) of DDR2 (without signal peptide and transmembrane domain, designated DR) in insect cells, purified and characterized DR, hoping to use it as a specific antagonist of DDR2. By using Bac-To-Bac Expression System with a His tag, we successfully obtained the recombinant bacularvirus containing DDR2 ECD, purified it and characterized its function. The soluble fraction of DR was about 12% of the total fused protein. After chromatographic purification, DR with 92% purity was obtained. Competitive inhibition assay demonstrated that DR blocked the binding between DDR2 and natural DDR2 receptors on NIH3T3 and synovial cells. Results of RT-PCR, Western blotting, and gelatinase zymography showed that DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIH3T3 and RA synoviocytes stimulated by collagen II. For MMP-1, inhibition was displayed at the levels of mRNA and protein, whereas for MMP-2 it was at the level of protein. These findings suggested that the expressed DR inhibited the activity of natural DDR2 and relevant MMP-1 and MMP-2 expression in RA synoviocytes and NIH3T3 cells provoked by collagen II.     

The expression of gelatinase A (MMP-2) is required for normal development of zebrafish embryos

Gelatinase A, also called matrix metalloproteinase 2 (MMP-2), belongs to the matrix metalloproteinase (MMP) family. MMP-2 cleaves type IV collagen, denatured collagen (gelatin), and other extracellular matrix (ECM) components. MMP-2 has been reported to be involved in a number of biological and pathological processes, but previous studies have not indicated that its expression is essential for early embryogenesis. In the current study, we have utilized zebrafish as a developmental model to study the role of MMP-2 during embryogenesis. We have successfully isolated a zebrafish MMP-2 (zMMP-2) homologue showing over 80% identity and over 90% similarity to its human counterpart. In situ analysis showed that zMMP-2 was expressed as early as the one-cell stage implying a maternal origin during oogenesis, and embryos continued to express zMMP-2 through at least the 72-h stage of development. RT-PCR analysis confirmed the in situ expression pattern and gelatin zymography indicated that a metalloproteinase with the same gel mobility as vertebrate MMP-2 was present in zebrafish embryos. Injection of zMMP-2 antisense morpholino oligonucleotides into 1- to 4-cell embryos resulted in a truncated axis, monitored through 72 h of development indicating that this metalloproteinase plays an important role in zebrafish embryogenesis. Monpholino-induced alterations in development began to be observed at 12 h of embryogenesis based on morphological and axis marker studies. The results obtained in zebrafish are in contrast to murine knockout studies that indicate that MMP-2 does not have a major role in mouse embryogenesis.Edited by D. Tautz     

Generation of conditional knockout alleles for PDGF-C

PDGF-C is a newly identified member of the platelet-derived growth factor (PDGF) family, which is involved in multiple cellular functions by signaling through PDGF receptor (PDGFR)-alphaalpha and alphabeta dimers. PDGF-C deficiency is perinatal lethal due to the formation of cleft palate. To further characterize the cellular function of PDGF-C during both embryonic and postnatal development, we have generated two conditional alleles of the Pdgf-c gene in which two loxP sites flank exon 5. Global Cre-mediated excision of the floxed exon 5 in these alleles resulted in a complete loss of PDGF-C expression and caused embryonic defects identical to those previously described for the PDGF-C null embryos. These conditional alleles will therefore be the important genetic tools for dissecting the spatial and temporal roles of PDGF-C during development and in adult tissues. Furthermore, from this work, we have also described a simple approach for creating mouse conditional alleles in an efficient manner.     

Matrix metalloproteinase (MMP)-12 regulates MMP-9 expression in interleukin-1beta-treated articular chondrocytes

Limited information is available on the expression and role of matrix metalloproteinase (MMP)-12 in chondrocytes. We characterized the expression mechanism of MMP-12 and possible function in chondrocytes. Interleukin (IL)-1beta induced the expression and activation of MMP-12 in primary culture chondrocytes and cartilage explants via mitogen-activated protein (MAP) kinase signaling pathways. Among MAP kinases, extracellular signal-regulated kinase and p38 kinase are necessary for MMP-12 expression, whereas c-jun N-terminal kinase is required for the activation of MMP-12. The possibility that MMP-12 acts as a modulator of other MMP was examined. MMP-12 alone did not affect other MMP expressions. However, MMP-12 enhanced expression and activation of MMP-9 in the presence of IL-1beta. Our results indicate that IL-1beta in chondrocytes induces the expression and activation of MMP-12, which, in turn, augments MMP-9 expression and activation.     

Matrix metalloproteinase‐2 and ‐9 activities in human keloids,hypertrophic and atrophic scars: a pilot study

Proteolytic degradation of extracellular matrix is one of the principal features of cutaneous wound healing but little is known about the activities of gelatinases; matrix metalloproteinase‐2 (MMP‐2) and matrix metalloproteinase‐9 (MMP‐9) on abnormal scar formation. The aim of this study is to determine collagen levels and the gelatinase activities in tissue from hypertrophic scars, atrophic scars, keloids and donor skin in 36 patients and 14 donors. Gelatinase levels (proenzyme + active enzyme) were determined by ELISA and their activities by gelatin zymography. MMP‐9 activity was undetectable in gelatin zymography analysis. Pro‐MMP‐2 levels (median) were highest in normal skin group 53.58 (36.40–75.11) OD µg^?1 protein, while active MMP‐2 levels were highest in keloid group 52.53 (42.47–61.51) OD µg^?1 protein. The active/pro ratio was the highest in keloid group 0.97 followed by hypertrophic scar, normal skin and atrophic scar groups 0.69 > 0.54 > 0.48, respectively. According to results of our study, the two‐phase theory of the duration of hypertrophic scar and keloid formation can be supported by the data of tissue collagen and gelatinase analysis. This study is the first to relate scar formation relationship in regard to gelatinase activation ratio in a keloid, hypertrophic and atrophic scar patient group which is chosen appropriate in age and sex. Copyright © 2009 John Wiley & Sons, Ltd.     

Antifungal triazole derivative triadimefon induces ectopic maxillary cartilage by altering the morphogenesis of the first branchial arch

BACKGROUND: The triazole derivative, triadimefon (FON), induces branchial arch abnormalities in post-implantation rat embryos cultured in vitro, and cranio-facial malformations in mouse fetuses. Ectopic maxillary cartilage has been also described as a typical FON-related malformation. This work studies the morphogenesis of the ectopic cartilage in rat embryos and fetuses exposed in vivo to FON during the early postimplantation period. METHODS: Pregnant rats were treated with 0, 250, and 500 mg/kg FON on Day 9.5 of pregnancy (D9.5) and sacrificed at term (D20), during the early fetal period (D17) or at different embryogenetic periods (D10, D11, D12). The skeleton was examined after stain of bone and cartilage or of cartilage alone respectively at term or at D17. The neural crest cell (NCC) migration and compaction was investigated at D10 and D11 and the cranial nerve organization described at D12. RESULTS: Triadimefon is teratogenic in rats under the chosen experimental conditions. The malformations were at the level of the cranio-facial and axial skeleton at term and of the hindbrain nerves in embryos. A NCC abnormal migration and compaction was observed at the level of the first branchial arch: in FON-exposed embryos NCC were detected at the level of both maxillary and mandibular processes, whereas control embryos showed the immunostained tissue only at the level of the mandibular bud. CONCLUSIONS: The pathogenic pathway, proposed to explain the ectopic cartilage, is the displacement of part of the NCC-derived tissues at the maxillary region of the first branchial arch.     

白细胞介素1β抑制心肌成纤维细胞胶原合成并促进其分解

本文应用3H-胸腺嘧啶核苷(3H-thymidine, 3H-TdR)掺入法及3H-脯氨酸(3H-proline, 3H-Pro)掺入法观察白细胞介素1β(interleukin-1β, IL-1β)对Spague-Dawley乳鼠心肌成纤维细胞DNA及胶原合成的影响,并用明胶酶谱法和Western blot检测基质金属蛋白酶(matrix metalloproteinases, MMPs) MMP-2、 MMP-9活性及MMP-2和MMP-9蛋白表达,用RT-PCR检测MMP-2、 MMP-9的mRNA表达.结果显示:(1)0.1、1、10、100ng/mL的IL-1β作用于细胞24h后,各组3H-TdR掺入量明显较对照组低(P<0.05, P<0.01),同时3H-Pro掺入量明显降低(P<0.05, P<0.01);而0.01ng/mL的IL-1β作用于细胞后,对3H-TdR掺入量和3H-Pro掺入量无明显影响.(2)不同剂量(0.01～100ng/mL)的IL-1β均刺激MMP-2和MMP-9活性升高,并呈剂量依赖性.IL-1β增加MMP-2和MMP-9蛋白表达(P<0.05, P<0.01).(3)IL-1β(0.01～100ng/mL)刺激MMP-2和MMP-9 mRNA表达升高(P<0.05, P<0.01).以上结果表明,IL-1β通过减少心肌成纤维细胞的细胞分裂来降低胶原的合成,同时促进MMP-2和MMP-9的转录及转录后的表达来促进胶原的分解,提示其在心肌重塑过程中起一定作用.     

Gene expression in Xenopus laevis embryos after Triadimefon exposure

The triazole derivative Triadimefon (FON) is a systemic fungicide used to control powdery mildews, rusts, and other fungal pests. Some data have already been published about the teratogenic activity of this compound: craniofacial malformations were found in mouse, rat, and Xenopus laevis embryos exposed to FON. These alterations were correlated to defective branchial arch development possibly caused by abnormal neural crest cell (NCC) migration into the branchial mesenchyme. As the migration of NCCs is controlled by the HOX code and by an anteroposterior retinoic acid (RA) gradient, we analyzed the expression of CYP26, a key enzyme in RA metabolism, following FON exposure. The increased expression of this gene and the ability of citral (a RA inhibitor) to reduce the teratogenic effects of the fungicide support the notion that endogenous RA is involved in the mechanism of action of FON. Moreover, by in situ hybridization, we studied the effects of FON exposure at gastrula stage on the expression of some genes involved in craniofacial development, hindbrain patterning, and NCC migration. We observed abnormal localization of xCRABP, Hoxa2 and Xbap signal expression at the level of migrating NCC domains, whereas in the hindbrain, we did not find any alteration in Krox20 and Hoxa2 expression.     

MMP-14 mediated MMP-9 expression is involved in TGF-beta1-induced keratinocyte migration

The importance of expression of matrix metalloproteinase (MMP) in keratinocyte migration is well established, but its role remains unclear. Here we investigated the function of MMP-14 in TGF-beta1-induced keratinocyte migration. TGF-beta1 stimulated cell migration and the expression of MMP-2, -9 in HaCaT human keratinocyte cells. When we lowered MMP-14 mRNA with siRNA, cell migration, and MMP-9 expression decreased. Furthermore, the MMP-14 siRNA also reduced activation of JNK in response to TGF-beta1, and a JNK-specific inhibitor decreased both cell migration and MMP-9 expression. Taken together, these results suggest that TGF-beta1-induced HaCaT cell migration is mediated by MMP-14, which regulates MMP-9 expression via JNK signaling.     

Induction of matrix metalloproteinase MMP-9 (92-kDa gelatinase) by retinoic acid in human neuroblastoma SKNBE cells: relevance to neuronal differentiation

Retinoic acid (RA) has been shown to induce human neuroblastoma SKNBE cell differentiation into a neuronal phenotype. Whether this neuronal differentiation is associated with modulation of matrix gelatinase [matrix metalloproteinase (MMP)-2 and MMP-9] expression was investigated in SKNBE cell cultures exposed to RA for 14 days. Their differentiation into a neuronal phenotype was typified by neural cell adhesion molecule and growth-associated protein-43 expression. Gelatinase expression was assessed by gel zymography, quantitative RT-PCR, and immunocytochemistry. Neuronal markers were located in neurites and ganglion-like clusters of neuronal cells induced upon RA exposure. MMP-2 expression was constitutive and remained unchanged at both the mRNA and protein levels in response to RA, tumor necrosis factor-alpha (TNFalpha), or phorbol 12-myristate 13-acetate (PMA) treatment. In contrast, MMP-9 was inducible by RA, TNFalpha, or PMA. MMP-9 was progressively enhanced by RA as a function of time exposure until day 14. The addition of TNFalpha or PMA potentiated RA-induced MMP-9 expression with a synergic maximal effect at day 14 of RA exposure. Immunoreactive MMP-9 was located early in outgrowing neurites, but only at day 14 of RA exposure in extensive neuritic networks. Taken together, the correlation between the MMP-9 expression by SKNBE cells and the time scale of their differentiation into a neuronal phenotype allowed us to propose that MMP-9 could participate in the neurite growth process and cell migration and organization into ganglion-like clusters.     

MMP-2 expression precedes the final ripening process of the bovine cervix

Collagen is denatured in the gradual cervical ripening process during late pregnancy, already before the onset of final cervical ripening at parturition. Matrix Metallo Proteinases (MMPs) might be responsible for this process. To investigate the presence and potential function of MMPs at the different stages of the ripening process, serial cervical biopsies were obtained from 10 cows at Days 185 and 275 of pregnancy (approximately 5 days before calving), at parturition and at 30 days after parturition. The mRNA and protein expression of MMP-1, MMP-2, and MMP-9 and of the tissue inhibitors of MMPs (TIMP)-1 and TIMP-2 were semi-quantitatively determined using RT-PCR, respectively, zymography, Westernblot, and ELISA techniques and the localization of MMP-2 protein and presence of granulocytes by immunohistochemistry and Luna staining. At parturition compared to 185 days pregnancy the MMP-1 protein expression and the numbers of granulocytes were significantly increased by 3 and 26-fold respectively. MMP-2 mRNA and protein expression had already increased 2.5 (P < 0.05) and twofold (P < 0.05) at 5 days before parturition, prior to final ripening. At that time, MMP-2 was present in smooth muscle cells and extra cellular matrix. TIMP-1 mRNA expression was significantly increased at parturition and TIMP-2 mRNA expression peaked at 5 days before parturition. The increased expression of MMP-2 at 5 days before parturition, suggests that in the cow MMP-2 is responsible for collagen denaturation in the last part of gradual cervical ripening, while MMP-1 and MMP-9 are only active during the final cervical ripening process at parturition.     

Early genetic control of craniofacial development is affected by the in vitro exposure of rat embryos to the fungicide triadimefon

BACKGROUND: Previous published experiments reported that in vitro exposure of postimplantation rat embryos to the triazole fungicide triadimefon (FON) resulted in specific abnormalities at the branchial apparatus and that the sensitive period is restricted to the first 24 hr of culture and is associated with the abnormal expression of TGF family genes (some of a large panel of genes regulated by retinoic acid (RA) and involved in branchial arch morphogenesis). The aim of this study is the determination of the sensitive window to FON‐induced abnormalities during in vitro development and the evaluation of the expression of some genes controlled by RA and involved in early branchial arch morphogenesis (Gsc, Msx1, Msx2, Dlx1, Dlx2, Shh, Patched (the main Shh receptor)). METHODS: Rat embryos were exposed in vitro to the FON under condition known to be able to induce 100% of abnormal embryos (250 µ M) at different stages and examined after 48 hr of culture. The sensitive window for FON‐induced abnormalities was during the hours E9 h8.00 PM–E10 h8.00 AM. To evaluate the expression of selected genes, embryos exposed during the sensitive stages were processed to perform quantitative PCR after 18 and 24 hr of culture. RESULTS: FON was able to affect the expression of some genes in a stage‐specific manner: earlier embryos were characterized by the downregulation of Msx2 and Gsc, later embryos showed the downregulation of Gsc, Shh, and Patched. The obtained data suggest that FON‐induced abnormalities are mediated, at least in part, through the imbalance of the expression of RA‐related signals. Birth Defects Res (Part B) 92:77‐81, 2011. © 2011 Wiley‐Liss, Inc.     

Retinoic acid-induced developmental defects are mediated by RARbeta/RXR heterodimers in the pharyngeal endoderm

Fusion and hypoplasia of the first two branchial arches, a defect typically observed in retinoic acid (RA) embryopathy, is generated in cultured mouse embryos upon treatment with BMS453, a synthetic compound that exhibits retinoic acid receptor beta (RARbeta) agonistic properties in transfected cells. By contrast, no branchial arch defects are observed following treatment with synthetic retinoids that exhibit RARalpha or RARgamma agonistic properties. The BMS453-induced branchial arch defects are mediated through RAR activation, as they are similar to those generated by a selective pan-RAR agonist, are prevented by a selective pan-RAR antagonist and cannot be mimicked by exposure to a pan-RXR agonist alone. They are enhanced in the presence of a pan-RXR agonist, and cannot be generated in Rarb-null embryos. Furthermore, they are accompanied, in the morphologically altered region, by ectopic expression of Rarb and of several other direct RA target genes. Therefore, craniofacial abnormalities characteristic of the RA embryopathy are mediated through ectopic activation of RARbeta/RXR heterodimers, in which the ligand-dependent activity of RXR is subordinated to that of RARbeta. Endodermal cells lining the first two branchial arches respond to treatment with the RARbeta agonist, in contrast to neural crest cells and ectoderm, which suggests that a faulty endodermal regionalization is directly responsible for RA-induced branchial arch dysmorphologies. Additionally, we provide the first in vivo evidence that the synthetic RARbeta agonist BMS453 exhibits an antagonistic activity on the two other RAR isotypes.     

Paradigmatic identification of MMP-2 and MT1-MMP activation systems in cardiac fibroblasts cultured as a monolayer

Activations of MMP-2 and membrane type 1-matrix metalloproteinase (MT1-MMP) have been correlated with cell migration, a key cellular event in the wound healing and tissue remodeling. We have previously demonstrated furin-dependent MMP-2 and MT1-MMP activations induced by type I collagen in cardiac fibroblasts. To understand mechanistic aspects of the regulation of MMP-2 and MT1-MMP activations by potential non-matrix factor(s) in cardiac fibroblasts, in the present study, we examined the effects of various agents including concanavalin A (ConA), a proteolytic phenotype-producing agent. We showed that treatment of cells with ConA activated pro-MMP-2, and that this activation concurred with elevated levels of cellular MT1-MMP and TIMP-2. The presence of active MT1-MMP and 43 and 36 kDa processed forms of MT1-MMP in a fraction of intracellular proteins prepared from ConA-treated cells suggests the possible internalization of differential forms of MT1-MMP. The appearance of 36 kDa processed form of MT1-MMP in conditioned media prepared from ConA-treated cells indicates the possible extracellular release of the further processed MT1-MMP fragment. Inhibition of furin in ConA-treated cells attenuated pro-MT1-MMP processing and the cellular TIMP-2 level, plus it reduced cell-released active MMP-2 in a time-dependent manner. These results suggest the involvement of furin in the ConA-induced activations of MT1-MMP and MMP-2. Furthermore, the existence of furin inhibitor-insensitive pro- and active MMP-2 species associated with ConA-treated cells implies that a mechanism independent of furin may perhaps account for the binding of the MMP-2 species to the cells. Supplementary material for this article can be found at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/94/suppmat_guo.tif.     

Correlation of HSP110 expression with all-trans retinoic acid-induced apoptosis

In a previous study, we observed the strong expression of a stress protein of the HSP100/Clp family (HSP110) in apoptotic mesectodermal cells during early mouse facial development. In the present study, we describe the strong expression of the same HSP110 in mesectodermal cells undergoing apoptosis after all-trans retinoic acid (RA) administration. We used a teratological model known to increase cell deaths mainly in the first and second branchial arches during mammalian cephalogenesis: the treatment of E9 mouse embryos with all-trans RA, which results in craniofacial malformations comparable to those that characterize mandibulofacial dysostosis in man. Pregnant NMRI mice were treated with 60 mg/kg body weight of all-trans RA, given orally on day 9 of gestation; embryos were taken 4, 12 or 24 hr after RA administration. The apoptotic pattern of RA-induced cell deaths was confirmed using the dUTP biotin nick-end labeling (TUNEL) method and transmission electron microscopy (TEM). HSP110 expression was detected using an immunohistochemical approach. The increase in the number of TUNEL-positive cells and HSP110-positive cells after all-trans RA administration was quantified in the first branchial arch using a computerized method. Twelve hours after RA administration, the increase in the number of HSP110-positive cells is greater than the increase in the number of TUNEL-positive cells. Twenty-four hours after RA administration, only TUNEL-positive cells remain strong in number. We suggest that HSP110 expression could represent a biochemical event of apoptotic cell death induced by RA, associated with early stages of the apoptotic process. In order to find out if HSP110 expression resulted from neosynthesis, we performed in situ hybridization, which demonstrated that the expression of HSP110 occurred at the level of mRNA.