Differential Regulation of Cell Type-specific Apoptosis by Stromelysin-3: A POTENTIAL MECHANISM VIA THE CLEAVAGE OF THE LAMININ RECEPTOR DURING TAIL RESORPTION IN XENOPUS LAEVIS*

Matrix metalloproteinases (MMPs) have been extensively studied because of their functional attributes in development and diseases. However, relatively few in vivo functional studies have been reported on the roles of MMPs in postembryonic organ development. Amphibian metamorphosis is a unique model for studying MMP function during vertebrate development because of its dependence on thyroid hormone (T3) and the ability to easily manipulate this process with exogenous T3. The MMP stromelysin-3 (ST3) is induced by T3, and its expression correlates with cell death during metamorphosis. We have previously shown that ST3 is both necessary and sufficient for larval epithelial cell death in the remodeling intestine. To investigate the roles of ST3 in other organs and especially on different cell types, we have analyzed the effect of transgenic overexpression of ST3 in the tail of premetamorphic tadpoles. We report for the first time that ST3 expression, in the absence of T3, caused significant muscle cell death in the tail of premetamorphic transgenic tadpoles. On the other hand, only relatively low levels of epidermal cell death were induced by precocious ST3 expression in the tail, contrasting what takes place during natural and T3-induced metamorphosis when ST3 expression is high. This cell type-specific apoptotic response to ST3 in the tail suggests distinct mechanisms regulating cell death in different tissues. Furthermore, our analyses of laminin receptor, an in vivo substrate of ST3 in the intestine, suggest that laminin receptor cleavage may be an underlying mechanism for the cell type-specific effects of ST3.The extracellular matrix (ECM),³ the dynamic milieu of the cell microenvironment, plays a critical role in dictating the fate of the cell. The cross-talk between the cell and ECM and the timely catabolism of the ECM are crucial for tissue remodeling during development (1). Matrix metalloproteinases (MMPs), extrinsic proteolytic regulators of the ECM, mediate this process to a large extent. MMPs are a large family of Zn²⁺-dependent endopeptidases potentially capable of cleaving the extracellular as well as nonextracellular proteins (2–9). The MMP superfamily includes collagenases, gelatinases, stromelysins, and membrane-type MMPs based on substrate specificity and domain organization (2–4). MMPs have been implicated to influence a wide range of physiological and pathological processes (10–13). The roles of MMPs appear to be very complex. For example, MMPs have been suggested to play roles in both tumor promotion and suppression (13–19). Unfortunately, relatively few functional studies have been carried out in vivo, especially in relation to the mechanisms involved during vertebrate development.Amphibian metamorphosis presents a fascinating experimental model to study MMP function during postembryonic development. A unique and salient feature of the metamorphic process is the absolute dependence on the signaling of thyroid hormone (20–23). This makes it possible to prevent metamorphosis by simply inhibiting the synthesis of endogenous T3 or to induce precocious metamorphosis by merely adding physiological levels of T3 in the rearing water of premetamorphic tadpoles. Gene expression screens have identified the MMP stromelysin-3 (ST3) as a direct T3 response gene (24–27). Expression studies have revealed a distinct spatial and temporal ST3 expression profile in correlation with metamorphic event, especially cell death (25, 28–31). Organ culture studies on intestinal remodeling have directly substantiated an essential role of ST3 in larval epithelial cell death and ECM remodeling (32). Furthermore, precocious expression of ST3 alone in premetamorphic tadpoles through transgenesis is sufficient to induce ECM remodeling and larval epithelial apoptosis in the tadpole intestine (33). Thus, ST3 appears to be necessary and sufficient for intestinal epithelial cell death during metamorphosis.ST3 was first isolated as a breast cancer-associated gene (34), and unlike most other MMPs, ST3 is secreted as an active protease through a furin-dependent intracellular activation mechanism (35). Like many other MMPs, ST3 is expressed in a number of pathological processes, including most human carcinomas (11, 36–40), as well as in many developmental processes in mammals (10, 34, 41–43), although the physiological and pathological roles of ST3 in vivo are largely unknown in mammals. Interestingly, compared with other MMPs, ST3 has only weak activities toward ECM proteins in vitro but stronger activities against non-ECM proteins like α1 proteinase inhibitor and IGFBP-1 (44–46). Although ST3 may cleave ECM proteins strongly in the in vivo environment, these findings suggest that the cleavage of non-ECM proteins is likely important for its biological roles. Consistently, we have recently identified a cell surface receptor, laminin receptor (LR) as an in vivo substrate of ST3 in the tadpole intestine during metamorphosis (47–49). Analyses of LR expression and cleavage suggest that LR cleavage by ST3 is likely an important mechanism by which ST3 regulates the interaction between the larval epithelial cells and the ECM to induce cell death during intestinal remodeling (47, 48).Here, to investigate the role of ST3 in the apoptosis in other tissues during metamorphosis and whether LR cleavage serves as a mechanism for ST3 to regulate the fate of different cell types, we have analyzed the effects of precocious expression of ST3 in premetamorphic tadpole tail. The tail offers an opportunity to examine the effects of ST3 on different cell types. The epidermis, the fast and slow muscles, and the connective tissue underlying the epidermis in the myotendinous junctions and surrounding the notochord constitute the major tissue types in tail (50). Even though death is the destiny of all these cell types, it is not clear whether they all die through similar or different mechanisms. Microscopic and histochemical analyses have shown that at least the muscle and epidermal cells undergo T3-dependent apoptosis during metamorphosis (23, 29, 51, 52). To study whether ST3 regulates apoptosis of these two cell types, we have made use of the transgenic animals that express a transgenic ST3 under the control of a heat shock-inducible promoter (33). We show that whereas extensive apoptosis is present in both the epidermis and muscles during natural as well as T3-induced metamorphosis, transgenic expression of ST3 induces cell death predominantly in the muscles. Furthermore, we show that LR is expressed in the epidermis and connective tissue but not in muscles of the tadpole tail. More importantly, LR cleavage products are present in the tail during natural metamorphosis but not in transgenic tadpoles overexpressing ST3. These results suggest that ST3 has distinct effects on the epidermis and muscles in the tail, possibly because of the tissue-specific expression and function of LR.     

Activin A Suppresses Osteoblast Mineralization Capacity by Altering Extracellular Matrix (ECM) Composition and Impairing Matrix Vesicle (MV) Production

During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP¹) activity in extracellular compartments, ECM and matrix vesicles. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts matrix vesicle production was deficient containing very low expression of annexin proteins. ECM enhanced human mesenchymal stem cell osteogenic development and mineralization. This osteogenic enhancement was significantly decreased when human mesenchymal stem cells were cultured on ECM produced under activin A treatment. These findings demonstrate that activin A targets the ECM maturation phase of osteoblast differentiation resulting ultimately in the inhibition of mineralization. ECM proteins modulated by activin A are not only determinant for bone mineralization but also possess osteoinductive properties that are relevant for bone tissue regeneration.The quality of bone tissue is determined by the balanced action of the anabolic bone cells, the osteoblasts, and their catabolic counterparts, the osteoclasts. This process of bone remodeling occurs throughout life and can be influenced by a wide variety of molecules, having ultimately an impact on the quality of bone (1, 2). Activins and inhibins are members of the TGF-β superfamily with predominant antagonistic effects in their classically known target tissues, such as in gonadotropin producing cells in the pituitary and their role in reproduction (3, 4). Like other TGF-β member, activins elicit biological responses by binding to type I and II serine/threonine kinase receptors at the cell surface. Upon ligand binding, signaling is further transduced in the cytoplasm by phosphorylated Smad protein complexes that once in the nucleus regulate gene expression. This signaling pathway is highly complex because of crosstalk between different ligands (Activins, BMPs, TGF-β) binding to multiple serine/threonine kinase receptors that activate different Smad proteins signaling to the nucleus. Activin is known to signal using type II receptors ACVR2A or ACVR2B and the type I receptor ACVRIB (shared with BMPs) activating Smad2 and 3 proteins (shared with TGF-β). Inhibins exert their inhibitory effects on activin by competitive binding to the activin receptors in the presence of betaglycan. This signaling regulates a wide array of biological activities from cell proliferation, differentiation to tumor development and endocrine signaling (5, 6) in many cell lineages like hematopoietic (7, 8) and monocyte/macrophage (9, 10). Several consequences of these reproductive hormones, especially those of activin A, are also described in relation to bone metabolism. Activin A is present in bone tissue (11, 12) affecting both osteoclasts and osteoblasts. While having a consistent pro-osteoclastogenic effect (9, 13), the activin A impact on osteoblast differentiation is more controversial (see (14) for review) Several reports support a stimulatory effect of activin A on osteoblast differentiation and mineralization in vitro and in vivo (9, 15, 16). On the other hand, two different studies, using rat and human bone formation models, have demonstrated that activin A treatment has a coherent inhibitory influence on osteogenesis leading to significant reduction of the mineralization capacity (11, 17). These opposing effects of activin A on osteoblastogenesis may simply reflect species differences, however, it may be also driven by heterogeneity of the used cell model or the stage of osteoblast differentiation (14). Nevertheless, a negative role of activin A in bone formation is also supported by other in vivo studies in mice and primates in which blockage of activin signaling resulted in increased bone mass (18, 19). Moreover, transgenic mice overexpressing human inhibin A showed increased bone formation (20).The extracellular compartment is crucial for bone because it determines most of the bone quality properties (21, 22), including its strength, stability, and integrity. Interestingly, a mature extracellular matrix (ECM) is characterized by the capacity to mineralize even in the absence of further osteoblast activity (11, 23). This biomineralization process is complex and not fully elucidated but it is thought to be started within MVs (24). Osteoblasts in bone and other cells in mineralization competent tissues, such as cartilage (25), tendon (26), teeth (27), and calcifying vasculature (28) produce and release from their plasma membrane these vesicles with diameters ranging between 50 and 200 nm. It is inside these membrane-enclosed particles that first crystals of mineral are formed and grow, before the vesicle membrane is permeated and the mineral crystallization advances into the ECM (29, 30). In this context, proteins that can mobilize calcium and inorganic phosphate (Pi), the backbone of the hydroxyapatite crystals present in bone, are of utmost importance. P_i donor proteins found in MVs include alkaline phosphatase (ALP) and inorganic pyrophosphatases (31) whereas the annexin family of proteins is postulated to be crucial for calcium influx into the vesicles (32–34).In this study we investigated the inhibitory effect of activin A on human mesenchymal stem cells (hMSC) derived osteoblast differentiation and mineralization. We have previously shown that in human osteoblast cultures activin A influences the expression of many ECM genes altering ECM maturity (11). Thus, we focused our analysis on extracellular environment changes, namely the ECM and matrix vesicles (MVs). The characterization of these compartments was done using the state-of-the-art quantitative proteomics tools including SILAC metabolic labeling and mass spectrometry. Furthermore, the importance of ECM composition for osteoblast differentiation was also determined.     

Heparan Sulfate Proteoglycans Are Receptors for the Cell-surface Trafficking and Biological Activity of Transglutaminase-2

Transglutaminase type 2 (TG2) is both a protein cross-linking enzyme and a cell adhesion molecule with an elusive unconventional secretion pathway. In normal conditions, TG2-mediated modification of the extracellular matrix modulates cell motility, proliferation and tissue repair, but under continuous cell insult, higher expression and elevated extracellular trafficking of TG2 contribute to the pathogenesis of tissue scarring. In search of TG2 ligands that could contribute to its regulation, we characterized the affinity of TG2 for heparan sulfate (HS) and heparin, an analogue of the chains of HS proteoglycans (HSPGs). By using heparin/HS solid-binding assays and surface plasmon resonance we showed that purified TG2 has high affinity for heparin/HS, comparable to that for fibronectin, and that cell-surface TG2 interacts with heparin/HS. We demonstrated that cell-surface TG2 directly associates with the HS chains of syndecan-4 without the mediation of fibronectin, which has affinity for both syndecan-4 and TG2. Functional inhibition of the cell-surface HS chains of wild-type and syndecan-4-null fibroblasts revealed that the extracellular cross-linking activity of TG2 depends on the HS of HSPG and that syndecan-4 plays a major but not exclusive role. We found that heparin binding did not alter TG2 activity per se. Conversely, fibroblasts deprived of syndecan-4 were unable to effectively externalize TG2, resulting in its cytosolic accumulation. We propose that the membrane trafficking of TG2, and hence its extracellular activity, is linked to TG2 binding to cell-surface HSPG.Transglutaminase type 2 (TG2,² EC 2.3.2.13) is the most widespread member of a large family of enzymes that catalyze the Ca²⁺-dependent post-translational modification of proteins leading to intra- or intermolecular Nϵ(γ-glutamyl)lysine bonds (1, 2). Unlike other family members, TG2 is uniquely exported through a yet to be elucidated non-conventional pathway. Once secreted, TG2 finds in the extracellular compartment the ideal conditions of high Ca²⁺ and low GTP concentration for the activation of its intrinsic transamidation activity (cross-linking) (2, 3). Intracellularly, GTP binding suppresses the Ca²⁺-dependent cross-linking activity and determines the additional GTPase activity of TG2 (4, 5), which is responsible for signal transduction (6). Once externalized, TG2 remains tightly bound to the cell surface and to the extracellular matrix (ECM) (7, 8), and it is rarely found free in the conditioned medium, unless overexpressed by cell transfection (9).Extracellular TG2 activity is involved in the cross-linking of the ECM, conferring resistance to matrix metalloproteinase and promoting cell-matrix interactions via cross-linking of fibronectin (FN) and collagen (1, 7, 11, 12). TG2 has an additional non-enzymatic role in the matrix as an integrin-β₁ co-receptor (8) by supporting RGD-independent cell adhesion to FN (8, 13, 14).Extracellular cross-linking and TG2-mediated adhesion facilitate the repair process in many tissue compartments (1, 2, 15, 16). On the other hand, uncontrolled cross-linking as a consequence of chronic cell insult and secretion of TG2 has been implicated in a number of pathological conditions, including kidney, liver, and pulmonary fibrosis (17–20).Understanding how TG2 is exported and targeted to the cell surface is critical for limiting its cellular secretion and extracellular action. Although a key trigger for TG2 export is cell stress (2, 21, 22), TG2 is not unspecifically released, because extracellular trafficking occurs in the absence of leakage of intracellular components and cells remain viable (23). We know that TG2 requires the tertiary structure of its active site region to be secreted (9); moreover, TG2 is acetylated on the N terminus (24), a process reported to affect membrane targeting of non-conventional secreted proteins (25). Two main binding partners for TG2, FN and integrin-β1, have both been attributed a possible role in the transport of TG2 to the cell surface (8, 26). FN was shown to co-localize with TG2 once released (26), and integrin-β1 to co-associate with TG2 in cells induced to differentiate (8).TG2 has also long been known to have some affinity for heparin (27, 28), a highly sulfated analogue of heparan sulfate (HS) glycosaminoglycan chains, which are abundant constituents of the cell surface/ECM. HS chains are linear polysaccharides consisting of alternating N-acetylated or N-sulfated glucosamine units (GlcNAc or GlcNS), and uronic acids (glucuronic acid GlcA or iduronic acid IdoA residues) (29), which only exist covalently bound to the core protein of cell-surface proteoglycans (syndecans and glypicans) and secreted proteoglycans (29). Heparin binding is a property common to many ECM proteins (29), but the level of affinity has never been established for TG2, which makes it difficult to estimate the real biological significance of this interaction. Heparan sulfate proteoglycans (HSPG) bind ECM ligands through the HS chains, influencing their biological activity, trafficking, and secretion. Among the HSPG subfamilies, the syndecans act as co-receptors for both ECM components and soluble ligands (30), and syndecan-4 has overlapping roles with extracellular TG2 in wound healing and fibrosis (31, 32). In this study, we show that TG2 has a surprisingly high affinity for heparin and HS, raising the hypothesis that HSPG are involved in its biological activity. We demonstrate that HSPGs are essential for the transamidating activity of TG2 at the cell surface and that syndecan-4 acts as a receptor for TG2, which is involved in the trafficking and cell-surface localization, and thus activity of TG2.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Identification of a Novel HtrA1-susceptible Cleavage Site in Human Aggrecan: EVIDENCE FOR THE INVOLVEMENT OF HtrA1 IN AGGRECAN PROTEOLYSIS IN VIVO

Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIα1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV³⁵⁶) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV³⁵⁶ neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.The mammalian high-temperature requirement A (HtrA) family of serine proteases is defined by a characteristic trypsin-like serine protease domain and one or two C-terminal PDZ domains. Four mammalian HtrA proteins have been identified to date, HtrA1–4. HtrA1 (also called PRSS11) is a ubiquitously expressed extracellular serine protease which contains a signal sequence for secretion, an insulin-like growth factor (IGF)²-binding protein domain, and a Kazal-type serine protease inhibitor domain in addition to the serine protease domain and one C-terminal PDZ domain (1). HtrA1 has been implicated in the progression of several pathologies including age-related macular degeneration, cancer, Alzheimer disease, rheumatoid arthritis, and osteoarthritis (OA) (2–10). HtrA1 has also been shown to inhibit osteoblast mineralization (11).Expression of HtrA1 has been found to be elevated in articular cartilage in association with OA (5). In addition, HtrA1 levels are up-regulated in murine cartilage after experimentally induced joint damage (6). The physiological role of HtrA1 in OA disease progression as well as in other pathologies is unclear. Preliminary studies using in vitro digestion assays suggest that HtrA1 might be capable of digesting cartilage extracellular matrix (ECM) proteins such as fibromodulin, cartilage oligomeric matrix protein (COMP), fibronectin, decorin, and aggrecan (6, 12, 13). Furthermore, it was recently reported that elevated levels of HtrA1 protein (∼7-fold above normal) are present in synovial fluids obtained from OA patients and that fibronectin fragments generated by HtrA1 cleavage induced the expression of catabolic enzymes such as matrix metalloproteinases-1 (MMP-1) and MMP-3 in synovial fibroblasts (4). HtrA1 has also been shown to modulate multiple signaling pathways in vitro. It binds to transforming growth factor-β family proteins including transforming growth factor-β1 and bone morphogenetic proteins 2 and 4 and inhibits signaling mediated by these factors (14, 15). In addition, HtrA1 has been shown to cleave IGF-binding protein-5 and possibly regulate signaling mediated by IGF (16). These findings suggest that the protease HtrA1 may play a physiological role in cartilage during OA.Articular cartilage is made up of chondrocytes surrounded by the ECM comprised mainly of the proteoglycan, aggrecan, and type II collagen. During normal homeostasis there is a dynamic balance between anabolic activities such as proteoglycan synthesis as well as catabolic activities in which the ECM is destroyed. When the catabolic activities of proteases, such as MMPs and aggrecanases, offset new matrix synthesis, focal degradation and loss of articular cartilage occurs, resulting in the development of OA. In some in vitro digestion studies, we and others have shown degradation of aggrecan by recombinant HtrA1 (6, 12, 13). In the present study we set out to examine the physiological relevance of aggrecan cleavage by HtrA1 in OA disease progression.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

An In-solution Ultrasonication-assisted Digestion Method for Improved Extracellular Matrix Proteome Coverage

Epithelial cell behavior is coordinated by the composition of the surrounding extracellular matrix (ECM); thus ECM protein identification is critical for understanding normal biology and disease states. Proteomic analyses of ECM proteins have been hindered by the insoluble and digestion-resistant nature of ECM. Here we explore the utility of combining rapid ultrasonication- and surfactant-assisted digestion for the detailed proteomics analysis of ECM samples. When compared with traditional overnight digestion, this optimized method dramatically improved the sequence coverage for collagen I, revealed the presence of hundreds of previously unidentified proteins in Matrigel, and identified a protein profile for ECM isolated from rat mammary glands that was substantially different from that found in Matrigel. In a three-dimensional culture assay to investigate epithelial cell-ECM interactions, mammary epithelial cells were found to undergo extensive branching morphogenesis when plated with mammary gland-derived matrix in comparison with Matrigel. Cumulatively these data highlight the tissue-specific nature of ECM composition and function and underscore the need for optimized techniques, such as those described here, for the proteomics characterization of ECM samples.Extracellular matrix (ECM)¹ is a critical component of the tissue microenvironment. ECM plays a pivotal role in embryonic stem cell development and differentiation (1, 2) as well as many physiological (3) and pathological processes, including cancer progression (4, 5). Cell regulation by ECM has been studied with high frequency in recent years (7, 8). However, our ability to globally characterize ECM composition both in vitro and in vivo has been severely limited because of several unique attributes of ECM proteins such as high molecular weight glycans and the presence of covalent protein cross-links (6, 9, 10). Traditional proteomics approaches have proven to be ineffective for the identification of ECM proteins as demonstrated by the fact that collagens, despite being the most abundant protein in mammals, are significantly underrepresented in tissue-based proteomics data sets.Ultrasonication has long been used for the digestion of bioorganic materials to allow for maximal and reproducible extraction and hence the accurate identification of small molecule and inorganic analytes (11). More recently, Capelo et al. (12) have used ultrasonic energy to catalyze tryptic digestion of proteins for subsequent mass spectrometry-based identification. Here we sought to determine whether this method could be optimized to prepare ECM samples for mass spectrometry-based analysis. For method development, we used rat tail collagen as a representative ECM protein for which current proteomics approaches have proven relatively unsuccessful. Type I collagen is defined as a right-handed triple helix heterotrimer comprising two identical α1 chains and one α2 chain that form a fibrillar network (6). The physical properties of the triple helical structure render the protein resistant to proteasch as trypsin (9). In this work, we focused our efforts on developing a digestion approach that improves our ability to perform proteomics analysis on a type I collagen preparation and then used this method to identify the protein composition of EHS murine chondrosarcoma matrix (10), herein referred to as Matrigel, and a matrix preparation from rat mammary tissue.In this study, we developed a digestion approach suitable for a two-dimensional liquid chromatography-tandem mass spectrometry-based analysis of ECM proteins. Our digestion approach involves three cycles of ultrasonication for rapid initial trypsin digestion followed by overnight digestion using an acid-labile surfactant. This approach resulted in significant improvement in collagen peptide identification and the identification of numerous ECM proteins previously uncharacterized in Matrigel and in mammary tissue. The application of our ECM-optimized ultrasonic assisted trypsin digestion method is anticipated to significantly advance the identification of tissue- and disease state-specific ECM proteins.     

Biochemical Characterization and Function of Complexes Formed by Hyaluronan and the Heavy Chains of Inter-��-inhibitor (HC��HA) Purified from Extracts of Human Amniotic Membrane

Clinically, amniotic membrane (AM) suppresses inflammation, scarring, and angiogenesis. AM contains abundant hyaluronan (HA) but its function in exerting these therapeutic actions remains unclear. Herein, AM was extracted sequentially with buffers A, B, and C, or separately by phosphate-buffered saline (PBS) alone. Agarose gel electrophoresis showed that high molecular weight (HMW) HA (an average of ∼3000 kDa) was predominantly extracted in isotonic Extract A (70.1 ± 6.0%) and PBS (37.7 ± 3.2%). Western blot analysis of these extracts with hyaluronidase digestion or NaOH treatment revealed that HMW HA was covalently linked with the heavy chains (HCs) of inter-α-inhibitor (IαI) via a NaOH-sensitive bond, likely transferred by the tumor necrosis factor-α stimulated gene-6 protein (TSG-6). This HC·HA complex (nHC·HA) could be purified from Extract PBS by two rounds of CsCl/guanidine HCl ultracentrifugation as well as in vitro reconstituted (rcHC·HA) by mixing HMW HA, serum IαI, and recombinant TSG-6. Consistent with previous reports, Extract PBS suppressed transforming growth factor-β1 promoter activation in corneal fibroblasts and induced mac ro phage apo pto sis. However, these effects were abolished by hyaluronidase digestion or heat treatment. More importantly, the effects were retained in the nHC·HA or rcHC·HA. These data collectively suggest that the HC·HA complex is the active component in AM responsible in part for clinically observed anti-inflammatory and anti-scarring actions.Hyaluronan (HA)⁴ is widely distributed in extracellular matrices, tissues, body fluids, and even in intracellular compartments (reviewed in Refs. 1 and 2). The molecular weight of HA ranges from 200 to 10,000 kDa depending on the source (3), but can also exist as smaller fragments and oligosaccharides under certain physiological or pathological conditions (1). Investigations over the last 15 years have suggested that low M_r HA can induce the gene expression of proinflammatory mediators and proangiogenesis, whereas high molecular weight (HMW) HA inhibits these processes (4–7).Several proteins have been shown to bind to HA (8) such as aggrecan (9), cartilage link protein (10), versican (11), CD44 (12, 13), inter-α-inhibitor (IαI) (14, 15), and tumor necrosis factor-α stimulated gene-6 protein (TSG-6) (16, 17). IαI consists of two heavy chains (HCs) (HC1 and HC2), both of which are linked through ester bonds to a chondroitin sulfate chain that is attached to the light chain, i.e. bikunin. Among all HA-binding proteins, only the HCs of IαI have been clearly demonstrated to be covalently coupled to HA (14, 18). However, TSG-6 has also been reported to form stable, possibly covalent, complexes with HA, either alone (19, 20) or when associated with HC (21).The formation of covalent bonds between HCs and HA is mediated by TSG-6 (22–24) where its expression is often induced by inflammatory mediators such as tumor necrosis factor-α and interleukin-1 (25, 26). TSG-6 is also expressed in inflammatory-like processes, such as ovulation (21, 27, 28) and cervical ripening (29). TSG-6 interacts with both HA (17) and IαI (21, 24, 30–33), and is essential for covalently transferring HCs on to HA (22–24). The TSG-6-mediated formation of the HC·HA complex has been demonstrated to play a crucial role in female fertility in mice. The HC·HA complex is an integral part of an expanded extracellular “cumulus” matrix around the oocyte, which plays a critical role in successful ovulation and fertilization in vivo (22, 34). HC·HA complexes have also been found at sites of inflammation (35–38) where its pro- or anti-inflammatory role remain arguable (39, 40).Immunostaining reveals abundant HA in the avascular stromal matrix of the AM (41, 42).⁵ In ophthalmology, cryopreserved AM has been widely used as a surgical graft for ocular surface reconstruction and exerts clinically observable actions to promote epithelial wound healing and to suppress inflammation, scarring, and angiogenesis (for reviews see Refs. 43–45). However, it is not clear whether HA in AM forms HC·HA complex, and if so whether such an HC·HA complex exerts any of the above therapeutic actions. To address these questions, we extracted AM with buffers of increasing salt concentration. Because HMW HA was found to form the HC·HA complex and was mainly extractable by isotonic solutions, we further purified it from the isotonic AM extract and reconstituted it in vitro from three defined components, i.e. HMW HA, serum IαI, and recombinant TSG-6. Our results showed that the HC·HA complex is an active component in AM responsible for the suppression of TGF-β1 promoter activity, linkable to the scarring process noted before by AM (46–48) and by the AM soluble extract (49), as well as for the promotion of macrophage death, linkable to the inflammatory process noted by AM (50) and the AM soluble extract (51).