Bromodomain-containing-protein-4 and cyclin-dependent-kinase-9 inhibitors interact synergistically in vitro and combined treatment reduces post-traumatic osteoarthritis severity in mice.

OBJECTIVE: Joint injury rapidly induces expression of primary response genes (PRGs), which activate a cascade of secondary genes that destroy joint tissues and initiate post-traumatic osteoarthritis (PTOA). Bromodomain-containing-protein-4 (Brd4) and cyclin-dependent-kinase-9 (CDK9) cooperatively control the rate-limiting step of PRG transactivation, including pro-inflammatory genes. This study investigated whether Brd4 and CDK9 inhibitors suppress inflammation and prevent PTOA development in vitro and in a mouse PTOA model. METHODS: The effects of Brd4 and CDK9 inhibitors (JQ1 and Flavopiridol) on PRG and associated secondary damage were rigorously tested in different settings. Short-term effects of inflammatory stimuli (IL-1ß, IL-6, TNF) on human chondrocyte PRG expression were assessed by RT-PCR and microarray after 5-h. We quantified glycosaminoglycan release from IL-1ß-treated bovine cartilage explants after 3-6 days, and osteoarthritic changes in mice after ACL-rupture using RT-PCR (2-24hrs), in vivo imaging of MMP activity (24hrs), AFM-nanoindentation (3-7days), and histology (3days-4wks). RESULTS: Flavopiridol and JQ1 inhibitors act synergistically, and a combination of both almost completely prevented the activation of most IL-1ß-induced PRGs in vitro by microarray analysis, and prevented IL-1ß-induced glycosaminoglycan release from cartilage explants. Mice given the drug combination showed reduced IL-1ß and IL-6 expression, less in vivo MMP activity, and lower synovitis (1.5 vs 4.9) and OARSI scores (2.8 vs 6.0) than untreated mice with ACL-rupture. CONCLUSIONS: JQ1 and Flavopiridol work synergistically to reduce injury response after joint trauma, suggesting that targeting Brd4 and/or CDK9 could be a viable strategy for PTOA prevention and treatment of early OA.

Inhibition of early response genes prevents changes in global joint metabolomic profiles in mouse post-traumatic osteoarthritis.

OBJECTIVE: Although joint injury itself damages joint tissues, a substantial amount of secondary damage is mediated by the cellular responses to the injury. Cellular responses include the production and activation of proteases (MMPs, ADAMTSs, Cathepsins), and the production of inflammatory cytokines. The trajectory of cellular responses is driven by the transcriptional activation of early response genes, which requires Cdk9-dependent RNA Polymerase II phosphorylation. Our objective was to determine whether inhibition of cdk9-dependent early response gene activation affects changes in the joint metabolome. DESIGN: To model post-traumatic osteoarthritis, we subjected mice to non-invasive Anterior Cruciate Ligament (ACL)-rupture joint injury. Following injury, mice were treated with flavopiridol - a potent and selective inhibitor of Cdk9 kinase activity - to inhibit Cdk9-dependent transcriptional activation, or vehicle control. Global joint metabolomics were analyzed 1 h after injury. RESULTS: We found that injury induced metabolomic changes, including increases in Vitamin D3 metabolism, anandamide, and others. Inhibition of primary response gene activation immediately after injury largely prevented the global changes in the metabolomics profiles. Cluster analysis of joint metabolomes identified groups of injury-induced and drug-responsive metabolites. CONCLUSIONS: Metabolomic profiling provides an instantaneous snapshot of biochemical activity representing cellular responses. We identified two sets of metabolites that change acutely after joint injury: those that require transcription of primary response genes, and those that do not. These data demonstrate the potential for inhibition of early response genes to alter the trajectory of cell-mediated degenerative changes following joint injury, which may offer novel targets for cell-mediated secondary joint damage.

Inhibition of CDK9 prevents mechanical injury-induced inflammation, apoptosis and matrix degradation in cartilage explants.

Joint injury often leads to post-traumatic osteoarthritis (PTOA). Acute injury responses to trauma induce production of pro-inflammatory cytokines and catabolic enzymes, which promote chondrocyte apoptosis and degrade cartilage to potentiate PTOA development. Recent studies show that the rate-limiting step for transcriptional activation of injury response genes is controlled by cyclin-dependent kinase 9 (CDK9), and thus it is an attractive target for limiting the injury response. Here, we determined the effects of CDK9 inhibition in suppressing the injury response in mechanically-injured cartilage explants. Bovine cartilage explants were injured by a single compressive load of 30 % strain at 100 %/s, and then treated with the CDK9 inhibitor Flavopiridol. To assess acute injury responses, we measured the mRNA expression of pro-inflammatory cytokines, catabolic enzymes, and apoptotic genes by RT-PCR, and chondrocyte viability and apoptosis by TUNEL staining. For long-term outcome, cartilage matrix degradation was assessed by soluble glycosaminoglycan release, and by determining the mechanical properties with instantaneous and relaxation moduli. Our data showed CDK9 inhibitor markedly reduced injury-induced inflammatory cytokine and catabolic gene expression. CDK9 inhibitor also attenuated chondrocyte apoptosis and reduced cartilage matrix degradation. Lastly, the mechanical properties of the injured explants were preserved by CDK9 inhibitor. Our results provide a temporal profile connecting the chain of events from mechanical impact, acute injury responses, to the subsequent induction of chondrocyte apoptosis and cartilage matrix deterioration. Thus, CDK9 is a potential disease-modifying agent for injury response after knee trauma to prevent or delay PTOA development.

Non-invasive mouse models of post-traumatic osteoarthritis.

Animal models of osteoarthritis (OA) are essential tools for investigating the development of the disease on a more rapid timeline than human OA. Mice are particularly useful due to the plethora of genetically modified or inbred mouse strains available. The majority of available mouse models of OA use a joint injury or other acute insult to initiate joint degeneration, representing post-traumatic osteoarthritis (PTOA). However, no consensus exists on which injury methods are most translatable to human OA. Currently, surgical injury methods are most commonly used for studies of OA in mice; however, these methods may have confounding effects due to the surgical/invasive injury procedure itself, rather than the targeted joint injury. Non-invasive injury methods avoid this complication by mechanically inducing a joint injury externally, without breaking the skin or disrupting the joint. In this regard, non-invasive injury models may be crucial for investigating early adaptive processes initiated at the time of injury, and may be more representative of human OA in which injury is induced mechanically. A small number of non-invasive mouse models of PTOA have been described within the last few years, including intra-articular fracture of tibial subchondral bone, cyclic tibial compression loading of articular cartilage, and anterior cruciate ligament (ACL) rupture via tibial compression overload. This review describes the methods used to induce joint injury in each of these non-invasive models, and presents the findings of studies utilizing these models. Altogether, these non-invasive mouse models represent a unique and important spectrum of animal models for studying different aspects of PTOA.

In vivo fluorescence reflectance imaging of protease activity in a mouse model of post-traumatic osteoarthritis.

OBJECTIVE: Joint injuries initiate a surge of inflammatory cytokines and proteases that contribute to cartilage and subchondral bone degeneration. Detecting these early processes in animal models of post-traumatic osteoarthritis (PTOA) typically involves ex vivo analysis of blood serum or synovial fluid biomarkers, or histological analysis of the joint. In this study, we used in vivo fluorescence reflectance imaging (FRI) to quantify protease, matrix metalloproteinase (MMP), and Cathepsin K activity in mice following anterior cruciate ligament (ACL) rupture. We hypothesized that these processes would be elevated at early time points following joint injury, but would return to control levels at later time points. DESIGN: Mice were injured via tibial compression overload, and FRI was performed at time points from 1 to 56 days after injury using commercially available activatable fluorescent tracers to quantify protease, MMP, and cathepsin K activity in injured vs uninjured knees. PTOA was assessed at 56 days post-injury using micro-computed tomography and whole-joint histology. RESULTS: Protease activity, MMP activity, and cathepsin K activity were all significantly increased in injured knees relative to uninjured knees at all time points, peaking at 1-7 days post-injury, then decreasing at later time points while still remaining elevated relative to controls. CONCLUSIONS: This study establishes FRI as a reliable method for in vivo quantification of early biological processes in a translatable mouse model of PTOA, and provides crucial information about the time course of inflammation and biological activity following joint injury. These data may inform future studies aimed at targeting these early processes to inhibit PTOA development.

The effect of noggin interference in a rabbit posterolateral spinal fusion model.

STUDY DESIGN: Noggin protein levels and spinal fusion rates were compared in a rabbit model after application of siRNA against BMP antagonist noggin in paraspinal muscle. OBJECTIVE: To test whether endogenous BMPs are sufficient to form bone in the absence of their antagonists, using noggin siRNA to interrupt the negative feedback loop on endogenous BMP within the paraspinal muscles in rabbits. Unused Posterolateral lumbar fusion is a standard surgical treatment for many spinal disorders, yet even under ideal conditions the rate of non-fusion approaches 25 %. BMPs are effective in promoting bone formation, and are inhibited by antagonists such as noggin. We have previously shown that in this model, endogenous BMPs are present and endogenous BMP antagonist noggin is strongly increased during spinal fusion. Previous studies have found that noggin siRNA enhanced spinal fusion in combination with supra-physiological amounts of exogenous BMP; however, the effect of the siRNA alone remains unknown. METHODS: A posterolateral intertransverse rabbit lumbar fusion was utilized, as established by Boden et al. SiRNA against noggin was electroporated into paraspinal muscle to determine its effect on fusion. Outcome measures included noggin protein expression, and assessment of spinal fusion at 6 weeks. RESULTS: SiRNAs were effective in reducing overexpressed noggin in vitro. Noggin protein was successfully knocked down in vivo for the initial 7 days in our rabbit model and returned to detectable levels by 4 weeks and to normal levels by 6 weeks. The overall fusion rate was not significantly enhanced compared to established controls from our earlier work (Tang et al.). CONCLUSIONS: Early noggin suppression does not appear to enhance the BMP activity sufficiently to significantly affect final fusion rates in our model.

Musculoskeletal changes following non-invasive knee injury using a novel mouse model of post-traumatic osteoarthritis.

OBJECTIVE: Post-traumatic osteoarthritis (PTOA) is a common consequence of traumatic joint injury, with 50% of anterior cruciate ligament (ACL) rupture patients developing PTOA within 10-20 years. Currently accepted mouse models of PTOA initiate symptoms using various methods, none of which faithfully mimic clinically-relevant injury conditions. In this study we characterize a novel non-invasive mouse model of PTOA that injures the ACL with a single load of tibial compression overload. We utilize this model to determine the time course of articular cartilage and subchondral bone changes following knee injury. DESIGN: Mice were euthanized 1, 3, 7, 14, 28, or 56 days after non-invasive knee injury. Knees were scanned using micro-computed tomography (µCT) in order to quantify subchondral trabecular bone, subchondral bone plate, and non-native bone formation (heterotopic ossification). Development of osteoarthritis (OA) was graded using the osteoarthritis research society international (OARSI) scale on histological sections of injured and uninjured knees. RESULTS: Following injury we observed a rapid loss of trabecular bone in injured knees compared to uninjured knees by 7 days post-injury, followed by a partial recovery of trabecular bone to a new steady state by 28 days post-injury. We also observed considerable non-native bone formation by 56 days post-injury. Grading of histological sections revealed deterioration of articular cartilage by 56 days post-injury, consistent with development of mild OA. CONCLUSIONS: This study establishes a novel mouse model of PTOA, and describes the time course of musculoskeletal changes following knee injury, helping to establish the window of opportunity for preventative treatment.

Comparative analysis with collagen type II distinguishes cartilage oligomeric matrix protein as a primary TGFß-responsive gene.

OBJECTIVE: This study aims to investigate the regulation of expression of Cartilage oligomeric matrix protein (COMP), which is predominately expressed by chondrocytes and functions to organize the extracellular matrix. Mutations in COMP cause two skeletal dysplasias: pseudoachondroplasia and multiple epiphyseal dysplasia. The mechanism controlling COMP expression during chondrocyte differentiation is still poorly understood. DESIGN: Primary human bone marrow-derived stem cells were induced to differentiate into chondrocyte by pellet cultures. We then compared the temporal expression of COMP with the well-characterized cartilage-specific Type II collagen (Col2a1), and their response to transforming growth factor (TGF)ß and Sox trio (Sox5, 6, and 9) stimulation. RESULTS: COMP and Col2a1 expression are differentially regulated by three distinct mechanisms. First, upregulation of COMP mRNA precedes Col2a1 by several days during chondrogenesis. Second, COMP expression is independent of high cell density but requires TGF-ß1. Induction of COMP mRNA by TGF-ß1 is detected within 2h in the absence of protein synthesis and is blocked by specific inhibitors of the TGFß signaling pathway; and therefore, COMP is a primary TFGß-response gene. Lastly, while Col2a1 expression is intimately controlled by the Sox trio, overexpression of Sox trio fails to activate the COMP promoter. CONCLUSION: COMP and Col2a1 expression are regulated differently during chondrogenesis. COMP is a primary response gene of TGFß and its fast induction during chondrogenesis suggests that COMP is suitable for rapidly accessing the chondrogenic potential of stem cells.

Dynamic compression of chondrocytes induces a Rho kinase-dependent reorganization of the actin cytoskeleton.

The signal transduction mechanisms in chondrocytes that recognize applied forces and elicit the appropriate biochemical cellular responses are not well characterized. A current theory is that the actin cytoskeleton provides an intracellular framework onto which mechanosensation mechanisms are assembled. The actin cytoskeleton is linked to the extracellular matrix at multi-protein complexes called focal adhesions, and evidence exists that focal adhesions mediate the conversion of external physical forces into appropriate biochemical signal transduction events. The Rho GTPases affect the arrangement of actin cytoskeletal structures, and enhance the formation of focal adhesions, which link the cytoskeleton to the extracellular matrix. A major effector pathway downstream of Rho is the activation of Rho kinase (ROCK), which phosphorylates and activates Lim kinase, which in turn phosphorylates and inhibits the actin-depolymerizing protein cofilin. The objectives of this study were threefold: first, to quantify the actin reorganization in response to dynamic compression of agarose-embedded chondrocytes. Second, to test whether Rho kinase is required for the actin cytoskeletal reorganization induced by dynamic compression. Third, to test whether dynamic compression alters the intracellular localization of Rho kinase and actin remodeling proteins in chondrocytes. Dynamic compression of agarose-embedded chondrocytes induced actin cytoskeletal remodeling causing a significant increase in punctate F-actin structures. Rho kinase activity was required for these cytoskeletal changes. Dynamic compression increased the amount of phosphorylated Rho kinase. The chemokine CCL20 and inducible nitric oxide synthase (iNOS) were the most highly upregulated genes by dynamic compression and this response was reduced by the Rho kinase inhibitors. In conclusion, we show that dynamic compression induces changes in the actin cytoskeleton of agarose-embedded chondrocytes, and we establish methodology to quantify these changes. Furthermore, we show that Rho kinase activity is required for this actin reorganization and gene expression induced by dynamic compression.

Interleukin-17 family and IL-17 receptors.

Interleukin-17 (IL-17) is a pro-inflammatory cytokine secreted by activated T-cells. Recently discovered related molecules are forming a family of cytokines, the IL-17 family. The prototype member of the family has been designated IL-17A. Due to recent advances in the human genome sequencing and proteomics five additional members have been identified and cloned: IL-17B, IL-17C, IL-17D, IL-17E and IL-17F. The cognate receptors for the IL-17 family identified thus far are: IL-17R, IL-17RH1, IL-17RL (receptor like), IL-17RD and IL-17RE. However, the ligand specificities of many of these receptors have not been established. The IL-17 signaling system is operative in disparate tissues such as articular cartilage, bone, meniscus, brain, hematopoietic tissue, kidney, lung, skin and intestine. Thus, the evolving IL-17 family of ligands and receptors may play an important role in the homeostasis of tissues in health and disease beyond the immune system. This survey reviews the biological actions of IL-17 signaling in cancers, musculoskeletal tissues, the immune system and other tissues.

Differential expression of multiple genes during articular chondrocyte redifferentiation.

Articular chondrocytes undergo a rapid change in phenotype and gene expression, termed dedifferentiation, when isolated from cartilage tissue and cultured on tissue culture plastic. On the other hand, "redifferentiation" of articular chondrocytes in suspension culture is characterized by decreased cellular proliferation and the reinitiation of synthesis of hyaline articular cartilage extracellular matrix molecules. The molecular triggers for these events have yet to be defined. Subtracted cDNA libraries representing genes involved in the early events of adult human articular chondrocyte redifferentiation were generated from human articular chondrocytes that were first cultured in monolayer, and subsequently transferred to suspension culture at 10(6) cells/ml for redifferentiation. Differential regulation of genes involved in cellular organization, nuclear structure, cellular growth regulation, and extracellular matrix deposition and remodeling were observed within 48 hr of this transfer. Many of these genes had not been previously identified in the chondrocyte differentiation pathway and a number of the isolated cDNAs did not have homologies to sequences in the public data banks. Genes involved in IL-6 signal transduction including acute phase response factor (APRF), Mn superoxide dismutase, and IL-6 itself were up-regulated in suspension culture. Membrane glycoprotein gp130, a component of the IL-6 receptor, was down-regulated. Other genes involved in cell polarity, cell adherence, apoptosis, and possibly TGF-beta signaling were differentially regulated. The differential regulation of the cytokine connective tissue growth factor (CTGF) during the early stages of articular chondrocyte redifferentiation, decreasing within 48 hours of transfer to suspension culture, was particularly interesting given its reported role in the stimulation of cellular proliferation. CTGF was highly expressed in proliferative monolayer culture, and then greatly reduced by redifferentiation in standard high-density suspension culture. When articular chondrocytes were seeded in suspension at low-density (10(4) cells/ml), however, high levels of CTGF were observed along with increased levels of mature articular cartilage extracellular matrix protein RNAs, such as type II collagen and aggrecan. Although the role of CTGF in articular cartilage biology remains to be elucidated, the results described here demonstrate the potential utility of subtractive hybridization in understanding the process of articular chondrocyte redifferentiation.

Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro.

Chondrocytes that were isolated from adult human articular cartilage changed phenotype during monolayer tissue culture, as characterized by a fibroblastic morphology and cellular proliferation. Increased proliferation was accompanied by downregulation of the cartilage-specific extracellular matrix proteoglycan, aggrecan, by cessation of type-II collagen expression, and by upregulation of type-I collagen and versican. This phenomenon observed in monolayer was reversible after the transfer of cells to a suspension culture system. The transfer of chondrocytes to suspension culture in alginate beads resulted in the rapid upregulation of aggrecan and type-II collagen and the downregulation of expression of versican and type-I collagen. Type-X collagen and osteopontin, markers of chondrocyte hypertrophy and commitment to endochondral ossification, were not expressed by adult articular chondrocytes cultured in alginate, even after 5 months. In contrast, type-X collagen was expressed within 2 weeks in a population of cells derived from a fetal growth plate. The inability of adult articular chondrocytes to express markers of chondrocyte hypertrophy has underscored the fundamental distinction between the differentiation pathways that lead to articular cartilage or to bone. Adult articular chondrocytes expressed only hyaline articular cartilage markers without evidence of hypertrophy.

Cartilage matrix protein forms a type II collagen-independent filamentous network: analysis in primary cell cultures with a retrovirus expression system.

Cartilage matrix protein (CMP) is expressed specifically in mature cartilage and consists of two von Willebrand factor A domains (CMP-A1 and CMP-A2) that are separated by an epidermal growth factor-like domain, and a coiled-coil tail domain at the carboxyl terminal end. We have shown previously that CMP interacts with type II collagen-containing fibrils in cartilage. In this study, we describe a type II collagen-independent CMP filament and we analyze the structural requirement for the formation of this type of filament. Recombinant wild-type CMP and two mutant forms were expressed in chick primary cell cultures using a retrovirus expression system. In chondrocytes, the wild-type virally encoded CMP is able to form disulfide bonded trimers and to assemble into filaments. Filaments also form with CMP whose Cys455 and Cys457 in the tail domain were mutagenized to prevent interchain disulfide bond formation. Therefore, intermolecular disulfide bonds are not necessary for the assembly of CMP into filaments. Both the wild-type and the double cysteine mutant also form filaments in fibroblasts, indicating that chondrocyte-specific factors are not required for filament formation. A truncated form of CMP that consists only of the CMP-A2 domain and the tail domain can form trimers but fails to form filaments, indicating that the deleted CMP-A1 domain and/or the epidermal growth factor domain are necessary for filament assembly but not for trimer formation. Furthermore, the expression of the virally encoded truncated CMP in chondrocyte culture disrupts endogenous CMP filament formation. Together these data suggest a role for CMP in cartilage matrix assembly by forming filamentous networks that require participation and coordination of individual domains of CMP.

Progression and recapitulation of the chondrocyte differentiation program: cartilage matrix protein is a marker for cartilage maturation.

During endochondral bone formation, chondrocytes in the cartilaginous anlage of long bones progress through a spatially and temporally regulated differentiation program before being replaced by bone. To understand this process, we have characterized the differentiation program and analyzed the relationship between chondrocytes and their extracellular environment in the regulation of the program. Our results indicate that, within an epiphyseal growth plate, the zone of proliferating chondrocytes is not contiguous with the zone of hypertrophic chondrocytes identified by the transcription of the type X collagen gene. We find that the postproliferative chondrocytes which make up the zone between the zones of proliferation and hypertrophy specifically transcribe the gene for cartilage matrix protein (CMP). This zone has been termed the zone of maturation. The identification of this unique population of chondrocytes demonstrates that the chondrocyte differentiation program consists of at least three stages. CMP translation products are present in the matrix surrounding the nonproliferative chondrocytes of both the zones of maturation and hypertrophy. Thus, CMP is a marker for postmitotic chondrocytes. As a result of the changes in gene expression during the differentiation program, chondrocytes in each zone reside in an extracellular matrix with a unique macromolecular composition. Chondrocytes in primary cell culture can proceed through the same differentiation program as they do in the cartilaginous rudiments. In culture, a wave of differentiation begins in the center of a colony and spreads to its periphery. The cessation of proliferation coincides with the appearance of CMP and eventually the cells undergo hypertrophy and synthesize type X collagen. These results reveal distinct switches at the proliferative-maturation transition and at the maturation-hypertrophy transition during chondrocyte differentiation and indicate that chondrocytes synthesize new matrix molecules and thus modify their preexisting microenvironment as differentiation progresses. However, when "terminally" differentiated hypertrophic chondrocytes are released from their surrounding environment and incubated in pellet culture, they stop type X collagen synthesis, resume proliferation, and reinitiate aggrecan synthesis. Eventually they cease proliferation and reinitiate CMP synthesis and finally type X collagen. Thus they are capable of recapitulating all three stages of the differentiation program in vitro. The data suggest a high degree of plasticity in the chondrocyte differentiation program and demonstrate that the progression and maintenance of this program is regulated, at least in part, by the extracellular environment which surrounds a differentiating chondrocyte during endochondral bone formation.

The role of coiled-coil alpha-helices and disulfide bonds in the assembly and stabilization of cartilage matrix protein subunits. A mutational analysis.

Cartilage matrix protein (CMP) exists as a disulfide-bonded homotrimer in the matrix of cartilage. Each monomer consists of two CMP-A domains that are separated by an epidermal growth factor-like domain. A heptad repeat-containing tail makes up the carboxyl-terminal domain of the protein. The secreted form of CMP contains 12 cysteine residues numbered C1 through C12. Two of these are in each of the CMP-A domains, six are in the epidermal growth factor-like domain, and two are in the heptad repeat-containing tail. Two major categories of mutant CMPs were generated to analyze the oligomerization process of CMP: a mini-CMP and a heptadless full-length CMP. The mini-CMP consists of the CMP-A2 domain and the heptad repeat-containing tail. In addition, a number of mutations affecting C9 through C12 were generated within the full-length, the mini-, and the heptad-less CMPs. The mutational analysis indicates that the heptad repeats are necessary for the initiation of CMP trimerization and that the two cysteines in the heptad repeat-containing tail are both necessary and sufficient to form intermolecular disulfide bonds in either full-length or mini-CMP. The two cysteines within a CMP-A domain form an intradomain disulfide bond.

Link protein is ubiquitously expressed in non-cartilaginous tissues where it enhances and stabilizes the interaction of proteoglycans with hyaluronic acid.

Link protein (LP) is an abundant protein of cartilage which stabilizes the interaction of aggrecan with hyaluronic acid (HA). In this study we report that LP is also present in a large number of embryonic non-cartilaginous tissues. We demonstrate, using RNase protection experiments, that the coding region of the LP mRNAs isolated from these tissues is identical to that present in cartilage. Furthermore, we show that the LP mRNAs are translated in non-cartilaginous tissues by the identification of LP polypeptides with monoclonal antibody 4B6/A5 in Western blots. LP is localized in the extracellular matrix of the mesoderm along the entire digestive tract and in the dermis of the embryonic skin as revealed by immunofluorescence analysis. Investigations on the interactions between LP and proteoglycans from skin and proventriculus demonstrate that LP can enhance the binding of proteoglycans from these tissues to HA. In addition, we find that the same proteoglycans bound to HA in the presence of LP are always more resistant to competition by soluble HA than in the absence of LP. Our results suggest that LP is involved in the stabilization of extracellular matrices of a wide variety of non-cartilaginous tissues.