Subantimicrobial Dose Doxycycline Worsens Chronic Arthritis-Induced Bone Microarchitectural Alterations in a Mouse Model: Role of Matrix Metalloproteinases?

Background: Rheumatoid arthritis (RA) is a chronic inflammatory joint disease hallmarked by irreversible damage of cartilage and bone. Matrix metalloproteinases (MMPs) involved in connective tissue remodeling play an important role in this process. Numerous MMPs have been examined in humans and animals, but their functions are still not fully understood. Therefore, we investigated the role of MMPs in the K/BxN serum-transfer model of RA with the broad-spectrum MMP inhibitor subantimicrobial dose doxycycline (SDD) using complex in vivo and in vitro methodolgy. Methods: Chronic arthritis was induced by repetitive i.p. injections of K/BxN serum in C57BL/6J mice. SDD was administered daily in acidified drinking water (0.5 mg/mL, 80 mg/kg) during the 30 days experimental period. Mechanonociceptive threshold of the paw was evaluated by aesthesiometry, grasping ability by grid test, arthritis severity by scoring, neutrophil myeloperoxidase activity by luminescence, vascular hyperpermeability and MMP activity by fluorescence in vivo imaging and the latter also by gelatin zymography, bone structure by micro-computed tomography (micro-CT). Plasma concentrations of doxycycline were determined by liquid chromatography-mass spectrometry analysis. Results: K/BxN serum induced significant inflammatory signs, mechanical hyperalgesia, joint function impairment, increased myeloperoxidase activity and vascular hyperpermeability. Significant increase of MMP activity was also observed both in vivo and ex vivo with elevation of the 57-60, 75, and 92 kDa gelatinolytic isoforms in the arthritic ankle joints, but neither MMP activity nor any above described functional parameters were influenced by SDD. Most importantly, SDD significantly reduced bone mineral density in the distal tibia and enhanced the Euler number in the ankle. Arthritis-induced microarchitectural alterations demonstrating increased irregularity and cancellous bone remodeling, such as increased Euler number was significantly elevated by SDD in both regions. Conclusion: We showed increase of various MMP activities in the joints by in vivo fluorescence imaging together with ex vivo zymography, and investigated their functional significance using the broad-spectrum MMP inhibitor SDD in the translational RA model. This is the first demonstration that SDD worsens arthritis-induced bone microarchitectural alterations, but it appears to be independent of MMP inhibition.

Investigation of retention mechanism of resorcinarene based cavitands by linear and nonlinear chromatography.

Cavitands are cavity-shaped cyclic oligomers and they can create host-guest interactions with various analytes, therefore they have applications in supramolecular chemistry, nanoscale reactions, chromatographic separations, drug encapsulation and delivery, biochemistry. The investigation of the chromatographic behavior of large molecules, such as resorcinarenes and cavitands is meager up to now. To understand the retention of resorcinarenes and cavitands in liquid chromatography, we studied their retention mechanism by the thermodynamic parameters calculated from the van't Hoff equation and by generation of an adsorption isotherm, which can describe the adsorption of the solute on the stationary phase surface. We compared the thermodynamics of the retention for cyclic oligomers in acetonitrile:water and methanol:water mobile phases. Furthermore, we determined the equilibrium adsorption isotherm by inverse method and we made an error analysis of the estimation obtained with the inverse method to ascertain the validity of the obtained isotherm parameters over a broader concentration range.

Matrilin-2, an extracellular adaptor protein, is needed for the regeneration of muscle, nerve and other tissues.

The extracellular matrix (ECM) performs essential functions in the differentiation, maintenance and remodeling of tissues during development and regeneration, and it undergoes dynamic changes during remodeling concomitant to alterations in the cell-ECM interactions. Here we discuss recent data addressing the critical role of the widely expressed ECM protein, matrilin-2 (Matn2) in the timely onset of differentiation and regeneration processes in myogenic, neural and other tissues and in tumorigenesis. As a multiadhesion adaptor protein, it interacts with other ECM proteins and integrins. Matn2 promotes neurite outgrowth, Schwann cell migration, neuromuscular junction formation, skeletal muscle and liver regeneration and skin wound healing. Matn2 deposition by myoblasts is crucial for the timely induction of the global switch toward terminal myogenic differentiation during muscle regeneration by affecting transforming growth factor beta/bone morphogenetic protein 7/Smad and other signal transduction pathways. Depending on the type of tissue and the pathomechanism, Matn2 can also promote or suppress tumor growth.

Retention behavior of resorcinarene-based cavitands on C8 and C18 stationary phases.

The understanding of the retention behavior of large molecules is an area of interest in liquid chromatography. Resorcinarene-based cavitands are cavity-shaped cyclic oligomers that can create host-guest interactions. We have investigated the chromatographic behavior of two types of cyclic tetramers as analytes in high-performance liquid chromatography. The experiments were performed at four different temperatures (15, 25, 35, 45°C) on two types of reversed stationary phases (C8 and C18 ) from two different manufacturers. We have found a huge difference between the retention of resorcinarenes and cavitands. In some cases, the retention factor of cavitands was even a hundred times larger than the retention factor of resorcinarenes. The retention of methylated derivates was two to four times larger compared to that of demethylated compounds on every column. The opposite retention behavior of the resorcinarenes and cavitands on the two types of stationary phases showed well the difference of the selectivity of the XTerra and BDS Hypersil columns. The retention mechanism was studied by the thermodynamic parameters calculated from the van't Hoff equation.

Upregulation of matrilin-2 expression in murine hepatic stellate cells during liver injury has no effect on fibrosis formation and resolution.

BACKGROUND & AIMS: Matrilins are a family of four oligomeric adaptor proteins whose functions in extracellular matrix assembly during pathophysiological events still need to be explored in more detail. Matrilin-2 is the largest family member and the only matrilin expressed in the naive liver. Several studies demonstrate that matrilin-2 interacts with collagen I, fibronectin or laminin-111-nidogen-1 complexes. All these matrix components get upregulated during hepatic scar tissue formation. Therefore, we tested whether matrilin-2 has an influence on the formation and/or the resolution of fibrotic tissue in the mouse liver. METHODS: Fibrosis was induced by infection with an adenovirus encoding cytochrome P450 2D6 (autoimmune liver damage) or by exposure to the hepatotoxin carbon tetrachloride. Fibrosis severity and matrilin-2 expression were assessed by immunohistochemistry. Hepatic stellate cells (HSCs) were isolated and analysed by immunocytochemistry and Transwell migration assays. RESULTS: Both autoimmune as well as chemically induced liver damage led to simultaneous upregulation of matrilin-2 and collagen I expression. Discontinuation of carbon tetrachloride exposure resulted in concomitant dissolution of both proteins. Activated HSCs were the source of de novo matrilin-2 expression. Comparing wild type and matrilin-2-deficient mice, no differences were detected in fibronectin and collagen I upregulation and resolution kinetics as well as amount or location of fibronectin and collagen I production and degradation. CONCLUSIONS: Our findings suggest that the absence of matrilin-2 has no effect on HSC activation and regression kinetics, synthetic activity, proliferative capacity, motility, or HSC apoptosis.

Mass-transfer properties of insulin on core-shell and fully porous stationary phases.

The mass-transfer properties of three superficially-porous packing materials, with 2.6 and 3.6µm particle and 1.9, 2.6, and 3.2µm inner core diameter, respectively, were investigated and compared with those of fully porous packings with similar particle properties. Several sources of band spreading in the chromatographic bed have been identified and studied according to the general rate model of chromatography. Besides the axial dispersion in the stream of the mobile phase, and the external mass transfer resistance, the intraparticle diffusion was studied in depth. The first absolute and the second central moments of the peaks of human insulin, over a wide range of mobile phase velocities were measured and used for the calculation of the mass-transfer coefficients. The experimental data were also analyzed using the stochastic or molecular dynamic model of Giddings and Eyring. The dissimilarities of the mass-transfer observed in the different columns were identified and evaluated.

Extracellular deposition of matrilin-2 controls the timing of the myogenic program during muscle regeneration.

Here, we identify a role for the matrilin-2 (Matn2) extracellular matrix protein in controlling the early stages of myogenic differentiation. We observed Matn2 deposition around proliferating, differentiating and fusing myoblasts in culture and during muscle regeneration in vivo. Silencing of Matn2 delayed the expression of the Cdk inhibitor p21 and of the myogenic genes Nfix, MyoD and Myog, explaining the retarded cell cycle exit and myoblast differentiation. Rescue of Matn2 expression restored differentiation and the expression of p21 and of the myogenic genes. TGF-ß1 inhibited myogenic differentiation at least in part by repressing Matn2 expression, which inhibited the onset of a positive-feedback loop whereby Matn2 and Nfix activate the expression of one another and activate myoblast differentiation. In vivo, myoblast cell cycle arrest and muscle regeneration was delayed in Matn2(-/-) relative to wild-type mice. The expression levels of Trf3 and myogenic genes were robustly reduced in Matn2(-/-) fetal limbs and in differentiating primary myoblast cultures, establishing Matn2 as a key modulator of the regulatory cascade that initiates terminal myogenic differentiation. Our data thus identify Matn2 as a crucial component of a genetic switch that modulates the onset of tissue repair.

Lack of Matrilin-2 favors liver tumor development via Erk1/2 and GSK-3ß pathways in vivo.

Matrilin-2 (Matn2) is a multidomain adaptor protein which plays a role in the assembly of extracellular matrix (ECM). It is produced by oval cells during stem cell-driven liver regeneration. In our study, the impact of Matn2 on hepatocarcinogenesis was investigated in Matn2(-/-) mice comparing them with wild-type (WT) mice in a diethylnitrosamine (DEN) model. The liver tissue was analyzed macroscopically, histologically and immunohistochemically, at protein level by Proteome Profiler Arrays and Western blot analysis. Matn2(-/-) mice exhibited higher susceptibility to hepatocarcinogenesis compared to wild-type mice. In the liver of Matn2(-/-) mice, spontaneous microscopic tumor foci were detected without DEN treatment. After 15 µg/g body weight DEN treatment, the liver of Matn2(-/-) mice contained macroscopic tumors of both larger number and size than the WT liver. In contrast with the WT liver, spontaneous phosphorylation of EGFR, Erk1/2 GSK-3α/ß and retinoblastoma protein (p-Rb), decrease in p21/CIP1 level, and increase in ß-Catenin protein expression were detected in Matn2(-/-) livers. Focal Ki-67 positivity of these samples provided additional support to our presumption that the lack of Matn2 drives the liver into a pro-proliferatory state, making it prone to tumor development. This enhanced proliferative capacity was further increased in the tumor nodules of DEN-treated Matn2(-/-) livers. Our study suggests that Matn2 functions as a tumor suppressor in hepatocarcinogenesis, and in this process activation of EGFR together with that of Erk1/2, as well as inactivation of GSK-3ß, play strategic roles.

Lipid droplet binding thalidomide analogs activate endoplasmic reticulum stress and suppress hepatocellular carcinoma in a chemically induced transgenic mouse model.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most frequent and aggressive primary tumor of the liver and it has limited treatment options. RESULTS: In this study, we report the in vitro and in vivo effects of two novel amino-trifluoro-phtalimide analogs, Ac-915 and Ac-2010. Both compounds bind lipid droplets and endoplasmic reticulum membrane, and interact with several proteins with chaperone functions (HSP60, HSP70, HSP90, and protein disulfide isomerase) as determined by affinity chromatography and resonant waveguide optical biosensor technology. Both compounds inhibited protein disulfide isomerase activity and induced cell death of different HCC cells at sub or low micromolar ranges detected by classical biochemical end-point assay as well as with real-time label-free measurements. Besides cell proliferation inhibiton, analogs also inhibited cell migration even at 250 nM. Relative biodistribution of the analogs was analysed in native tissue sections of different organs after administration of drugs, and by using fluorescent confocal microscopy based on the inherent blue fluorescence of the compounds. The analogs mainly accumulated in the liver. The effects of Ac-915 and Ac-2010 were also demonstrated on the advanced stages of hepatocarcinogenesis in a transgenic mouse model of N-nitrosodiethylamine (DEN)-induced HCC. Significantly less tumor development was found in the livers of the Ac-915- or Ac-2010-treated groups compared with control mice, characterized by less liver tumor incidence, fewer tumors and smaller tumor size. CONCLUSION: These results imply that these amino-trifluoro-phthalimide analogs could serve potent clinical candidates against HCC alone or in combination with dietary polyunsaturated fatty acids.

Altered expression of genes for Kir ion channels in dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a multifactorial disease characterized by left ventricular dilation that is associated with systolic dysfunction and increased action potential duration. The Kir2.x K⁺ channels (encoded by KCNJ genes) regulate the inward rectifier current (IK1) contributing to the final repolarization in cardiac muscle. Here, we describe the transitions in the gene expression profiles of 4 KCNJ genes from healthy or dilated cardiomyopathic human hearts. In the healthy adult ventricles, KCNJ2, KCNJ12, and KCNJ4 (Kir2.1-2.3, respectively) genes were expressed at high levels, while expression of the KCNJ14 (Kir2.4) gene was low. In DCM ventricles, the levels of Kir2.1 and Kir2.3 were upregulated, but those of Kir2.2 channels were downregulated. Additionally, the expression of the DLG1 gene coding for the synapse-associated protein 97 (SAP97) anchoring molecule exhibited a 2-fold decline with increasing age in normal hearts, and it was robustly downregulated in young DCM patients. These adaptations could offer a new aspect for the explanation of the generally observed physiological and molecular alterations found in DCM.

Hmgb1 can facilitate activation of the matrilin-1 gene promoter by Sox9 and L-Sox5/Sox6 in early steps of chondrogenesis.

The architectural high mobility group box 1 (Hmgb1) protein acts as both a nuclear and an extracellular regulator of various biological processes, including skeletogenesis. Here we report its contribution to the evolutionarily conserved, distinctive regulation of the matrilin-1 gene (Matn1) expression in amniotes. We previously demonstrated that uniquely assembled proximal promoter elements restrict Matn1 expression to specific growth plate cartilage zones by allowing varying doses of L-Sox5/Sox6 and Nfi proteins to fine-tune their Sox9-mediated transactivation. Here, we dissected the regulatory mechanisms underlying the activity of a conserved distal promoter element 1. We show that this element carries three Sox-binding sites, works as an enhancer in vivo, and allows promoter activation by the Sox5/6/9 chondrogenic trio. In early steps of chondrogenesis, declining Hmgb1 expression overlaps with the onset of Sox9 expression. Unlike repression in late steps, Hmgb1 overexpression in early chondrogenesis increases Matn1 promoter activation by the Sox trio, and forced Hmgb1 expression in COS-7 cells facilitates induction of Matn1 expression by the Sox trio. The conserved Matn1 control elements bind Hmgb1 and SOX9 with opposite efficiency in vitro. They show higher HMGB1 than SOX trio occupancy in established chondrogenic cell lines, and HMGB1 silencing greatly increases MATN1 and COL2A1 expression. Together, these data thus suggest a model whereby Hmgb1 helps recruit the Sox trio to the Matn1 promoter and thereby facilitates activation of the gene in early chondrogenesis. We anticipate that Hmgb1 may similarly affect transcription of other cartilage-specific genes.

Evolutionarily conserved, growth plate zone-specific regulation of the matrilin-1 promoter: L-Sox5/Sox6 and Nfi factors bound near TATA finely tune activation by Sox9.

To help uncover the mechanisms underlying the staggered expression of cartilage-specific genes in the growth plate, we dissected the transcriptional mechanisms driving expression of the matrilin-1 gene (Matn1). We show that a unique assembly of evolutionarily conserved cis-acting elements in the Matn1 proximal promoter restricts expression to the proliferative and prehypertrophic zones of the growth plate. These elements functionally interact with distal elements and likewise are capable of restricting the domain of activity of a pancartilaginous Col2a1 enhancer. The proximal elements include a Pe1 element binding the chondrogenic L-Sox5, Sox6, and Sox9 proteins, a SI element binding Nfi proteins, and an initiator Ine element binding the Sox trio and other factors. Sox9 binding to Pe1 is indispensable for functional interaction with the distal promoter. Binding of L-Sox5/Sox6 to Ine and Nfib to SI modulates Sox9 transactivation in a protein dose-dependent manner, possibly to enhance Sox9 activity in early stages of chondrogenesis and repress it at later stages. Hence, our data suggest a novel model whereby Sox and Nfi proteins bind to conserved Matn1 proximal elements and functionally interact with each other to finely tune gene expression in specific zones of the cartilage growth plate.

Comparison of the mass transfer in totally porous and superficially porous stationary phases in liquid chromatography.

The characterization of mass-transfer processes in a chromatographic column during a separation process is essential, since the influence of the mass-transfer kinetics on the shape of the chromatographic band profiles and on the efficiency of the separation is crucial. Several sources of mass transfer in a chromatographic bed have been identified and studied: the axial dispersion in the stream of mobile phase, the external mass-transfer resistance, intraparticle diffusion, and the kinetics of adsorption-desorption. We measured and compared the characteristics and performance of a new brand of shell particles and those of a conventional brand of totally porous silica particles. The shell stationary phase was made of 2.7-microm superficially porous particles (a 1.7-microm solid core is covered with a 0.5-microm-thick shell of porous silica). The other material consisted of totally porous particles of conventional 3.5-microm commercial silica. We measured the first and second central moments of the peaks of human insulin over a wide range of mobile phase velocities (from 0.02 to 1.3 mL/min) at 20 degrees C. The plate height equations were constructed and the axial dispersion, external mass transfer, as well as the intraparticle diffusion coefficients were calculated for the two stationary phases.

Expression of matrilin-2 in liver cirrhosis and hepatocellular carcinoma.

The recently described matrilin protein family is part of the extracellular matrix, their pathophysiological role as well as distribution in liver diseases, however, have not yet been studied. Considering that matrilins have been found to play role in cell growth and tissue remodeling, their possible involvement in carcinogenesis has been raised. The main objective of this study was to investigate the changes in matrilin-2 expression which is one of the main components of basement membranes. Thirty-five cases of surgically resected hepatocellular carcinomas, 35 corresponding surrounding liver tissues and 10 normal liver samples were used for the study. In 15 of 35 cases the tumor developed on the basis of cirrhosis. Matrilin-2 protein expression was detected in normal liver around bile ducts, portal blood vessels, while sinusoids were negative by immunohistochemistry. Cirrhotic surrounding tissue showed intensive matrilin-2 staining along the sinusoids. Tumorous neovasculature was found strongly positive by immunohistochemistry. No differences, however, were detected by morphometry regarding the amount of protein expression based on the grade of hepatocellular carcinomas. Real-time RT-PCR did not show significant differences in matrilin-2 mRNA expression between normal, cirrhotic and tumor samples. This suggests posttranslational modification of matrilin-2 manifesting in altered distribution in liver fibrosis. Our data indicate that matrilin-2 is a novel basement membrane component in the liver, which is synthetised during sinusoidal "capillarization" in cirrhosis and in hepatocellular carcinoma. This is the first report to describe the expression and distribution of matrilin-2 in human normal and cirrhotic liver as well as in hepatocellular carcinoma.

Expression of matrilin-2 in oval cells during rat liver regeneration.

The matrilins represent a new family of oligomeric proteins that are assumed to act as adapter molecules connecting other proteins and proteoglycans in the extracellular matrix. Matrilin-2, the largest member of the family, displays a broad tissue distribution. It incorporates into loose and dense connective tissue and becomes associated with some basement membranes. The aim of our study was to analyse the expression of matrilin-2 in two liver regeneration models and to identify its cellular origin. Liver regeneration was induced in rats by partial hepatectomy (PH) and by the 2-acetylaminofluorene (AAF)/partial hepatectomy (PH) experimental models. Formalin fixed, paraffin embedded tissue sections were used for immunohistochemistry applying a rabbit matrilin-2 polyclonal antibody. Matrilin-2 was detected in normal rat liver and partially hepatectomized liver in the portal area, but could not be demonstrated in the acini. Matrilin-2 mRNA expression was analysed by RT-PCR and in situ hybridization. In the AAF/PH model the oval cells but not the hepatocytes produced matrilin-2 mRNA. Increase in protein level in the AAF/PH regenerating liver model was demonstrated by Western blotting. The protein was present in the basement membrane zone around the tubules formed by oval cells. Our data show that hepatic oval cells produce matrilin-2, a novel ECM protein, suggesting that matrilin-2 is an important component of ECM during stem cell-driven liver regeneration.

Identification of differentially expressed genes in the developing antler of red deer Cervus elaphus.

Understanding the molecular mechanisms underlying bone development is a fundamental and fascinating problem in developmental biology, with significant medical implications. Here, we have identified the expression patterns for 36 genes that were characteristic or dominant in the consecutive cell differentiation zones (mesenchyme, precartilage, cartilage) of the tip section of the developing velvet antler of red deer Cervus elaphus. Two major functional groups of these genes clearly outlined: six genes linked to high metabolic demand and other five to tumor biology. Our study demonstrates the advantages of the antler as a source of mesenchymal markers, for distinguishing precartilage and cartilage by different gene expression patterns and for identifying genes involved in the robust bone development, a striking feature of the growing antler. Putative roles for "antler" genes that encode alpha-tropomyosine (tpm1), transgelin (tagln), annexin 2 (anxa2), phosphatidylethanolamine-binding protein (pebp) and apolipoprotein D (apoD) in intense but still controlled tissue proliferation are discussed.

Expression pattern of matrilins and other extracellular matrix proteins characterize distinct stages of cell differentiation during antler development.

Deer antler regeneration is a uniquely intense and complex process, which involves chondrogenic and intramembranous ossification. Cell differentiation in the developing antler of red deer, Cervus elaphus, was characterized with extracellular matrix markers. Expression of the four matrilin genes was monitored by immunohistochemistry and in situ hybridization and compared to cartilage markers collagen II and cartilage link protein, the bone component collagen I, and the endothelial basement membrane constituent laminin. The mesenchyme layer at the very tip of the velvet antler was enriched in link protein, indicative of the role of hyaluronan in apical morphogenesis. Matrilin-2, formerly described as a component of hard and soft connective tissue matrices, was identified here also as a marker of cells with high differentiation potential: it is expressed predominantly by mesenchyme cells, prechondrocytes and preosteoblasts. In addition to matrilin-3, documented as a component of the bony extracellular matrix, expression of the other three matrilin genes was observed in osteoprogenitor cells and osteoblasts. A layer of presumed osteoprogenitor cells, which surrounded the perivascular channels, expressed all four matrilins and collagen I. As a consequence, all four matrilins, including matrilin-1, previously detected in the skeleton only in cartilage, were found associated to collagen I-rich structures in a thin layer bordering the columns of hypertrophic chondrocytes. Cells with similar morphology and expression pattern were identified in the periosteum. Altogether all cell types of the chondrogenic and osteogenic lineage that expressed the four matrilins were in a separate study [Faucheux, C., Nicholls, B.M., Allen, S., Danks, J.A, Horton, M.A., Price, J.S., 2004. Recapitulation of the parathyroid hormone-related peptide-Indian hedgehog pathway in the regenerating deer antler. Dev. Dyn. 231, 88-97] positive for parathyroid hormone-related peptide and its receptor.

Highly conserved proximal promoter element harbouring paired Sox9-binding sites contributes to the tissue- and developmental stage-specific activity of the matrilin-1 gene.

The matrilin-1 gene has the unique feature that it is expressed in chondrocytes in a developmental stage-specific manner. Previously, we found that the chicken matrilin-1 long promoter with or without the intronic enhancer and the short promoter with the intronic enhancer restricted the transgene expression to the columnar proliferative chondroblasts and prehypertrophic chondrocytes of growth-plate cartilage in transgenic mice. To study whether the short promoter shared by these transgenes harbours cartilage-specific control elements, we generated transgenic mice expressing the LacZ reporter gene under the control of the matrilin-1 promoter between -338 and +67. Histological analysis of the founder embryos demonstrated relatively weak transgene activity in the developing chondrocranium, axial and appendicular skeleton with highest level of expression in the columnar proliferating chondroblasts and prehypertrophic chondrocytes. Computer analysis of the matrilin-1 genes of amniotes revealed a highly conserved Pe1 (proximal promoter element 1) and two less-conserved sequence blocks in the distal promoter region. The inverted Sox motifs of the Pe1 element interacted with chondrogenic transcription factors Sox9, L-Sox5 and Sox6 in vitro and another factor bound to the spacer region. Point mutations in the Sox motifs or in the spacer region interfered with or altered the formation of nucleoprotein complexes in vitro and significantly decreased the reporter gene activity in transient expression assays in chondrocytes. In vivo occupancy of the Sox motifs in genomic footprinting in the expressing cell type, but not in fibroblasts, also supported the involvement of Pe1 in the tissue-specific regulation of the gene. Our results indicate that interaction of Pe1 with distal DNA elements is required for the high level, cartilage- and developmental stage-specific transgene expression.

Mice lacking the extracellular matrix adaptor protein matrilin-2 develop without obvious abnormalities.

Matrilins are putative adaptor proteins of the extracellular matrix (ECM) which can form both collagen-dependent and collagen-independent filamentous networks. While all known matrilins (matrilin-1, -2, -3, and -4) are expressed in cartilage, only matrilin-2 and matrilin-4 are abundant in non-skeletal tissues. To clarify the biological role of matrilin-2, we have developed a matrilin-2-deficient mouse strain. Matrilin-2 null mice show no gross abnormalities during embryonic or adult development, are fertile, and have a normal lifespan. Histological and ultrastructural analyses indicate apparently normal structure of all organs and tissues where matrilin-2 is expressed. Although matrilin-2 co-localizes with matrilin-4 in many tissues, Northern hybridization, semiquantitative RT-PCR, immunohistochemistry and biochemical analysis reveal no significant alteration in the steady-state level of matrilin-4 expression in homozygous mutant mice. Immunostaining of wild-type and mutant skin samples indicate no detectable differences in the expression and deposition of matrilin-2 binding partners including collagen I, laminin-nidogen complexes, fibrillin-2 and fibronectin. In addition, electron microscopy reveals an intact basement membrane at the epidermal-dermal junction and normal organization of the dermal collagen fibrils in mutant skin. These data suggest that either matrilin-2 and matrilin-2-mediated matrix-matrix interactions are dispensable for proper ECM assembly and function, or that they are efficiently compensated by other matrix components including wild-type levels of matrilin-4.

Functional analysis of the regulatory regions of the matrilin-1 gene in transgenic mice reveals modular arrangement of tissue-specific control elements.

Matrilin-1 is a non-collagenous protein, which functions in the organization of the extracellular matrix by forming collagen-dependent and -independent filamentous networks. It is secreted primarily by chondrocytes in a characteristic spatial, temporal and developmental stage-specific pattern during skeletogenesis. As a first step to define the tissue- and site-specific regulatory regions of the chicken matrilin-1 gene in vivo, we generated transgenic mice harboring various promoter and intronic fragments fused to the LacZ reporter gene. Histological analysis of the transgene expression pattern during ontogenic development revealed specific X-gal staining in most primordial elements of endochondral bones of transgenic mouse lines carrying either the long promoter between -2011 and +67 or the intronic fragment with a short promoter between -338 and +1819. The cartilage-specific activity of the latter transgene, however, was accompanied with variable ectopic expression pattern in neural and other tissues depending on the site of integration. The presence of both promoter upstream and intronic elements was necessary for the high level transgene activity in all chondrogenic tissues and for the extraskeletal transgene expression pattern resembling the most to that of the chicken matrilin-1 gene, e.g. expression in the eye, and lack of expression in the diminishing notochord and nucleus pulposus. The activity of the transgenes was restricted to the columnar proliferating and pre-hypertrophic chondrocytes visualized by BrdU incorporation and distribution of phosphorylated Sox9, respectively. DNA elements between -2011 and -338 also mediated ectopic LacZ expression in cells of neural crest origin. These results suggest that an interplay of modularly arranged cartilage- and neural crest-specific DNA elements control the expression of the matrilin-1 gene. The dispersal of cartilage-specific elements in the promoter upstream and intronic regions shows similarity to the transcriptional regulation of the Col11a2 gene.