Regulation of thrombospondin secretion by cells in culture

Thrombospondin (TS), a 450,000 molecular weight glycoprotein, is released from α-granules of thrombin-activated platelets and is secreted and incorporated into the extracellular matrix by several cell types in culture. We have examined the effects of cell density and transformation on the production of TS in cell culture. The levels of TS, per cell, in the culture medium of endothelial cells, smooth muscle cells, and fibroblasts were greater at lower cell densities; in fibroblasts the levels of two other extracellular matrix proteins, fibronectin and collagen, were unaffected by cell density. Our evidence indicates that the higher levels of TS in the culture medium, determined for lower-density cells, were achieved by an increased secretion of the protein rather than by a reduction in degradation or incorporation into the extracellular matrix. TS production by normal and transformed Wl-38 fibroblasts was the same, although the fibronectin level in the culture medium of the transformed cells was substantially decreased. These findings suggest that the production of TS by cells in culture is regulated in a different fashion from that of fibronectin or collagen.     

Tenascin-C mRNA and Tenascin-C Protein Immunoreactivity Increase in Astrocytes after Activation by bFGF

Tenascin-C is an extracellular matrix glycoprotein with trophic and repulsive properties, involved in migratory processes in CNS. Previous reports demonstrated that this molecule is produced and secreted by astrocytes. Preliminary data on fibroblasts and astrocytes have suggested that bFGF may modulate tenascin-C expression. bFGF is a mitogenic growth factor, involved in cell differentiation and neovascularization. In the present study, we ex amined whether bFGF modulates the expression of tenascin-C in hippocampal astrocytes from newborn rats. Our results suggest that bFGF increases the production of tenascin-C by cultured hippocampal astrocytes. We found that both tenascin-C mRNA and protein immunoreactivity were increased after bFGF treatment. Our results also demonstrated that tenas cin-C polypeptides were secreted into the extracellular medium. In agreement with previous studies, we suggest that secreted tenascin-C is mainly the high molecular weight form. In addition, our results suggest that a cleavage of the high molecular weight form may occur in the extracellular medium causing production of proteolytic fragments, that may modify the biological properties of tenascin-C. The present results may be relevant to the understanding of lesion scarring and regeneration process.     

Proteomic Analysis of Human Dermal Fibroblast Conditioned Medium (DFCM)

Growth factors and extracellular matrix (ECM) proteins are involved in wound healing. Human dermal fibroblasts secrete wound-healing mediators in culture medium known as dermal fibroblast conditioned medium (DFCM). However, the composition and concentration of the secreted proteins differ with culture conditions and environmental factors. We cultured human skin fibroblasts in vitro using serum-free keratinocyte-specific media (EpiLife? Medium [KM1] and defined keratinocyte serum-free medium [KM2]) and serum-free fibroblast-specific medium (FM) to obtain DFCM-KM1, DFCM-KM2 and DFCM-FM, respectively. We identified and compared their proteomic profiles using bicinchoninic acid assay (BCA), 1-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography MS (LC-MS/MS). DFCM-KM1 and DFCM-KM2 had higher protein concentrations than DFCM-FM but not statistically significant. MALDI-TOF/TOF MS identified the presence of fibronectin, serotransferrin, serpin and serum albumin. LC-MS/MS and bioinformatics analysis identified 59, 46 and 58 secreted proteins in DFCM-KM1, DFCM-KM2 and DFCM-FM, respectively. The most significant biological processes identified in gene ontology were cellular process, metabolic process, growth and biological regulation. STRING® analysis showed that most secretory proteins in the DFCMs were associated with biological processes (e.g. wound healing and ECM organisation), molecular function (e.g. ECM binding) and cellular component (e.g. extracellular space). ELISA confirmed the presence of fibronectin and collagen in the DFCMs. In conclusion, DFCM secretory proteins are involved in cell adhesion, attachment, proliferation and migration, which were demonstrated to have potential wound-healing effects by in vitro and in vivo studies.     

Identification and Characterization of a Novel 38.5-Kilodalton Cell Surface Protein of Staphylococcus aureus with Extended-Spectrum Binding Activity for Extracellular Matrix and Plasma Proteins

The ability to attach to host ligands is a well-established pathogenic factor in invasive Staphylococcus aureus disease. In addition to the family of adhesive proteins bound to the cell wall via the sortase A (srtA) mechanism, secreted proteins such as the fibrinogen-binding protein Efb, the extracellular adhesion protein Eap, or coagulase have been found to interact with various extracellular host molecules. Here we describe a novel protein, the extracellular matrix protein-binding protein (Emp) initially identified in Western ligand blots as a 40-kDa protein due to its broad-spectrum recognition of fibronectin, fibrinogen, collagen, and vitronectin. Emp is expressed in the stationary growth phase and is closely associated with the cell surface and yet is extractable by sodium dodecyl sulfate. The conferring gene emp (1,023 nucleotides) encodes a signal peptide of 26 amino acids and a mature protein of a calculated molecular mass of 35.5 kDa. Using PCR, emp was demonstrated in all 240 S. aureus isolates of a defined clinical strain collection as well as in 6 S. aureus laboratory strains, whereas it is lacking in all 10 S. epidermidis strains tested. Construction of an allelic replacement mutant (mEmp50) revealed the absence of Emp in mEmp50, a significantly decreased adhesion of mEmp50 to immobilized fibronectin and fibrinogen, and restoration of these characteristics upon complementation of mEmp50. Emp expression was also demonstrable upon heterologous complementation of S. carnosus. rEmp expressed in Escherichia coli interacted with fibronectin, fibrinogen, and vitronectin in surface plasmon resonance experiments at a K(d) of 21 nM, 91 nM, and 122 pM, respectively. In conclusion, the biologic characterization of Emp suggests that it is a member of the group of secreted S. aureus molecules that interact with an extended spectrum of host ligands and thereby contribute to S. aureus pathogenicity.     

The role of human HtrA1 in arthritic disease

Human HtrA1 belongs to a widely conserved family of serine proteases involved in various aspects of protein quality control and cell fate. Although HtrA1 has been implicated in the pathology of several diseases, its precise biological functions remain to be established. Through identification of potential HtrA1 targets, studies presented herein propose that within the context of arthritis pathology HtrA1 contributes to cartilage degradation. Elevated synovial HtrA1 levels were detected in fluids obtained from rheumatoid and osteoarthritis patients, with synovial fibroblasts identified as a major source of secreted HtrA1. Mass spectrometry analysis of potential HtrA1 substrates within synovial fluids identified fibronectin as a candidate target, and treatment of fibronectin with recombinant HtrA1 led to the generation of fibronectin-degradation products that may be involved in cartilage catabolism. Consistently, treatment of synovial fibroblasts with HtrA1 or HtrA1-generated fibronectin fragments resulted in the specific induction of matrix metalloprotease 1 and matrix metalloprotease 3 expression, suggesting that HtrA1 contributes to the destruction of extracellular matrix through both direct and indirect mechanisms.     

Regulation of protein secretion in Chinese hamster ovary cells by cell cycle position and cell density : Plasminogen activator, procollagen and fibronectin

To study the relationship between cell growth control, cell contact, and protein secretion, we examined the production of plasminogen activator, procollagen, and fibronectin by Chinese hamster ovary (CHO) fibroblasts, both as a function of position in the cell cycle and as a function of cell density. CHO fibroblasts that were synchronized at hourly intervals throughout the cell cycle by mitotic selection in an automated roller bottle apparatus secreted plasminogen activator only during the G2 and M phases of the cell cycle (10–14 h after mitotic selection). Cell-associated plasminogen activator activity was variable during G1 and S, but was greatly reduced during G2 and M. In contrast, secretion of the connective tissue matrix proteins, procollagen and fibronectin, was controlled by cell density rather than by cell cycle position. Type III procollagen and fibronectin were secreted throughout the cell cycle with no pronounced variations. Type I procollagen was not secreted by cycling cells and was observed in confluent cultures only after 24–48 h. To correlate these changes in protein secretion patterns with cell shape and contact, we used scanning electron microscopy (SEM) to study the appearance of CHO cells after mitotic selection. Actively dividing cells retained a high proportion of rounded, ruffled, and blebbed cells during all phases of the cell cycle. Only with increased cell density in contact-inhibited confluent cultures did most cells begin to flatten and spread. Thus, secretion of and attachment to extracellular matrix did not occur in rapidly dividing cells, but appeared to require the increased cell-cell contact and spreading that accompanies contact inhibition of growth. On the other hand, increased secretion of plasminogen activator was directly related to cell division and may be part of a sequence of events that allows cells growing in culture to loosen extracellular attachments in preparation for rounding and cytokinesis.     

Cell-surface associated proteins of corneal fibroblasts: dissection with monoclonal antibodies

It is now generally accepted that the cell surface is involved in the interaction of the cells with the extracellular matrix. To identify and characterize cell-surface-associated components of corneal fibroblasts, several monoclonal antibodies were developed. Hybridomas were developed by fusing mouse myeloma cells SP2/OAg14 with spleen cells from mice immunized with membrane fractions of corneal fibroblasts grown in culture. Twenty-five hybridomas secreting monoclonal antibodies to cell-surface components were selected by an enzyme-linked immunosorbent assay using corneal fibroblasts grown in microtiter plates as the substrate. Immunohistochemical staining demonstrated that the antigenic determinants recognized by these antibodies were not present on corneal epithelial cells, but were present on skin fibroblasts. The antigenic determinants recognized by two of these antibodies, designated 10D2 and 716, were matrix components of the corneal stroma. Immunochemical characterization of the antigens was carried out by indirect precipitation of the radioactively labeled cellular proteins with the monoclonal antibodies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the precipitates. Four antibodies were able to precipitate antigens from cell extract in detectable amounts. Antibodies designated 5E2, 9G2, and 10D2 recognized antigens consisting of polypeptides of approximate molecular weights 105K and 110K, while antibody 716 recognized an antigen of 100K molecular weight. However, based on the tissue distribution and cell-surface distribution, these antibodies reacted with different antigenic determinants. The antigen recognized by 716 was also secreted by cells in culture but consisted of 220K and 200K polypeptide chains. It was tentatively identified as cellular fibronectin, based on the reaction of this antigen with polyclonal antibodies to plasma fibronectin.     

Matrix assembly sites for exogenous fibronectin are decreased on human fibroblasts after treatment with agents which increase intracellular cAMP

Studies with cultured fibroblasts have shown that plasma as well as cellular fibronectin can be organized into fibrillar structures and that this organization is mediated by sites at the cell surface. Treatment of human skin fibroblasts with cholera toxin resulted in a prompt decrease in the number of binding sites for 125I-labeled plasma fibronectin and a 125I-labeled 70-kDa amino-terminal fragment of fibronectin. This decrease was accompanied by less incorporation of labeled fibronectin into deoxycholate-insoluble extracellular matrix. Binding of 125I-fibronectin was also decreased in cultures treated with epinephrine, isoproterenol, or forskolin. These results, therefore, indicate that G proteins and the adenylate cyclase system are involved in regulation of fibronectin matrix assembly sites may be one mechanism whereby hormones or growth factors can modify extracellular matrix characteristics.     

Collagen VI deficiency affects the organization of fibronectin in the extracellular matrix of cultured fibroblasts.

Fibronectin is one of the main components of the extracellular matrix and associates with a variety of other matrix molecules including collagens. We demonstrate that the absence of secreted type VI collagen in cultured primary fibroblasts affects the arrangement of fibronectin in the extracellular matrix. We observed a fine network of collagen VI filaments and fibronectin fibrils in the extracellular matrix of normal murine and human fibroblasts. The two microfibrillar systems did not colocalize, but were interconnected at some discrete sites which could be revealed by immunoelectron microscopy. Direct interaction between collagen VI and fibronectin was also demonstrated by far western assay. When primary fibroblasts from Col6a1 null mutant mice were cultured, collagen VI was not detected in the extracellular matrix and a different pattern of fibronectin organization was observed, with fibrils running parallel to the long axis of the cells. Similarly, an abnormal fibronectin deposition was observed in fibroblasts from a patient affected by Bethlem myopathy, where collagen VI secretion was drastically reduced. The same pattern was also observed in normal fibroblasts after in vivo perturbation of collagen VI-fibronectin interaction with the 3C4 anti-collagen VI monoclonal antibody. Competition experiments with soluble peptides indicated that the organization of fibronectin in the extracellular matrix was impaired by added soluble collagen VI, but not by its triple helical (pepsin-resistant) fragments. These results indicate that collagen VI mediates the three-dimensional organization of fibronectin in the extracellular matrix of cultured fibroblasts.     

Hyaluronate binding proteins also bind to fibronectin, laminin and collagen

Small molecular weight proteins, isolated from the culture medium of embryonic chick heart fibroblasts and 3T3 cell lines by hyaluronate affinity chromatography, bind in order of apparent affinity, to hyaluronate, fibronectin, collagen and laminin. Such proteins isolated from the MSV-transformed 3T3 cell line bind in greater amounts to the nectins and hyaluronate than do similar proteins isolated from heart fibroblasts or 3T3 cells. These small hyaluronate binding proteins are immunologically distinct from other well characterized proteins such as laminin, fibronectin, bovine serum albumin and actin. Their relationship to other small, extracellular proteins and their possible role in structuring of extracellular matrix are discussed.     

The Effects of Age and the Expression of SPARC on Extracellular Matrix Production by Cardiac Fibroblasts in 3-D Cultures

Fibrillar collagen is the primary component of the cardiac interstitial extracellular matrix. This extracellular matrix undergoes dramatic changes from birth to adulthood and then into advanced age. As evidence, fibrillar collagen content was compared in sections from neonates, adult, and old hearts and was found to increase at each respective age. Cardiac fibroblasts are the principle cell type that produce and control fibrillar collagen content. To determine whether fibroblast production, processing, and deposition of collagen differed with age, primary cardiac fibroblasts from neonate, adult, and old mice were isolated and cultured in 3-dimensional (3D) fibrin gels. Fibroblasts from each age aligned in fibrin gels along points of tension and deposited extracellular matrix. By confocal microscopy, wild-type neonate fibroblasts appeared to deposit less collagen into fibrillar structures than fibroblasts from adults. However, by immunoblot analysis, differences in procollagen production and processing of collagen I were not detected in neonate versus adult fibroblasts. In contrast, fibroblasts from old mice demonstrated increased efficiency of procollagen processing coupled with decreased production of total collagen. SPARC is a collagen-binding protein previously shown to affect cardiac collagen deposition. Accordingly, in the absence of SPARC, less collagen appeared to be associated with fibroblasts of each age grown in fibrin gels. In addition, the increased efficiency of procollagen alpha 1(I) processing in old wild-type fibroblasts was not detected in old SPARC-null fibroblasts. Increased levels of fibronectin were detected in wild-type neonate fibroblasts over that of adult and old fibroblasts but not in SPARC-null neonate fibroblasts versus older ages. Immunostaining of SPARC overlapped with that of collagen I but not to that of fibronectin in 3D cultures. Hence, whereas increases in procollagen processing, influenced by SPARC expression, plausibly contribute to increased collagen deposition in old hearts, other cellular mechanisms likely affect differential collagen deposition by neonate fibroblasts.     

Regulation of fibronectin biosynthesis by glucocorticoids in human fibrosarcoma cells and normal fibroblasts

When treated with the synthetic glucocorticoid dexamethasone, HT1080 human fibrosarcoma cells show changes in morphology, adhesion, and the extracellular matrix. Dexamethasone treatment results in a tenfold increase in the rate of fibronectin biosynthesis in HT1080 cells and a twofold increase in untransformed, normal human fibroblasts. Maximal induction levels are attained within one cell generation, while decay of the response requires several cell cycles. Pulse-chase studies showed that most of the newly synthesized fibronectin is secreted into the medium. The glucocorticoid antagonist, RU-486, blocks the dexamethasone-induced changes but does not alter the basal rate of fibronectin production. Therefore, fibronectin biosynthesis appears to be controlled by two distinct mechanisms--one, regulating basal rates of fibronectin production, which is transformation-sensitive and glucocorticoid-independent; and another, which is mediated by the glucocorticoid receptor, resulting in elevated rates of fibronectin biosynthesis upon dexamethasone treatment both in normal fibroblasts and in HT1080 cells.     

Factor XIIIa-mediated cross-linking of fibronectin in fibroblast cell layers. Cross-linking of cellular and plasma fibronectin and of amino-terminal fibronectin fragments

Factor XIIIa cross-links plasma fibronectin as it is being assembled into the extracellular matrix of cultured human skin fibroblasts (Barry, E. L. R., and Mosher, D. F. (1988) J. Biol. Chem. 262, 10464-10469). We have further characterized this process. Fibroblasts were metabolically labeled with proline in the presence or absence of ascorbate and Factor XIIIa. Endogenous fibronectin in the extracellular matrix was cross-linked by Factor XIIIa. There was no evidence for cross-linking of collagenous proteins. Fibro-blast cell layers were incubated with iodinated 27-kDa heparin-binding or 70-kDa collagen- and heparin-binding amino-terminal fibronectin fragments. Factor XIIa cross-linked the fragments into high molecular weight aggregates. The amounts of cross-linked fragments reaches a steady state after 1 to 2 h, whereas intact fibronectin continues to be cross-linked for 24 h. When fibroblast cell layers were pulsed with iodinated fibronectin or amino-terminal fragments and Factor XIIIa was included in the chase media, the high molecular weight aggregates were formed in a step-wise manner. The smallest cross-linking steps were to high molecular weight extracellular matrix molecules forming approximately 270-, 300-, and 440-kDa complexes for the 27-kDa fragment, 70-kDa fragment, and intact fibronectin, respectively. When iodinated fibronectin was bound to fibroblast cell layers and chased into the matrix pool in the absence of Factor XIIIa, it could also be cross-linked into high molecular weight complexes when Factor XIIIa was added to the media. These results, therefore, indicate that both cellular and plasma fibronectin and amino-terminal fragments are cross-linked specifically by Factor XIIIa, that the cross-linking is probably to other fibronectin molecules rather than to collagenous proteins, and that both assembling and assembled fibronectin are substrates for Factor XIIIa.     

Extracellular Alix regulates integrin-mediated cell adhesions and extracellular matrix assembly

Alix (ALG-2-interacting protein X), a cytoplasmic adaptor protein involved in endosomal sorting and actin cytoskeleton assembly, is required for the maintenance of fibroblast morphology. As Alix has sequence similarity to adhesin in Entamoeba histolytica, and we observed that Alix is secreted, we determined whether extracellular Alix affects fibroblast morphology. Here, we demonstrate that secreted Alix is deposited on the substratum of non-immortalized WI38 fibroblasts. Antibody binding to extracellular Alix retards WI38 cell adhesion and spreading on fibronectin and vitronectin. Alix knockdown in WI38 cells reduces spreading and fibronectin assembly, and the effect is partially complemented by coating recombinant Alix on the cell substratum. Immortalized NIH/3T3 fibroblasts deposit less Alix on the substratum and have defects in α₅β₁-integrin functions. Coating recombinant Alix on the culture substratum for NIH/3T3 cells promotes α₅β₁-integrin-mediated cell adhesions and fibronectin assembly, and these effects require the aa 605–709 region of Alix. These findings demonstrate that a sub-population of Alix localizes extracellularly and regulates integrin-mediated cell adhesions and fibronectin matrix assembly.     

The activities of MMP‐9 and total gelatinase respond differently to substrate coating and cyclic mechanical stretching in fibroblasts and myoblasts

The current study was designed to investigate whether the activities of TGC (total gelatinase and collagenase) as well as MMP‐9 (matrix metalloproteinase‐9, gelatinase B) secreted by the cultured fibroblasts and myoblasts were influenced by the specific extracellular substrates and by cyclic mechanical strain. Fibroblasts (Rat 2) and myoblasts (C2C12) were cultured with either fibronectin, laminin or collagen type I for 24 h and applied with or without a biaxial deformation at 1 Hz using the Flexcell FX‐4000 system. MMP‐9 activity was increased in fibroblasts when the cells were in contact with fibronectin and laminin, while in myoblasts, enhanced activity of the secreted enzyme was only observed when collagen was present. TGC activity expressed from myoblasts was increased in cells growing on all three types of extracellular proteins in response to the mechanical stimulation, but in fibroblasts, such an increase was only observed in cells grown on the laminin coating. In summary, our data demonstrate that the activities of MMP‐9 synthesized by fibroblasts tend to be regulated by the specific extracellular protein the cells are in contact with, whereas the gelatinolytic actions of proteases produced by myoblasts are more responsive to the mechanical deformation.     

Regulation of cell surface tissue transglutaminase: effects on matrix storage of latent transforming growth factor-beta binding protein-1.

Using a cytochemical approach, we examined the role of tissue transglutaminase (tTgase, Type II) in the incorporation of latent TGF-beta binding protein-1 (LTBP-1) in the extracellular matrix of Swiss 3T3 fibroblasts in which tTgase expression can be modulated through a tetracycline-controlled promoter. Increased tTgase expression led to an increased rate of LTBP-1 deposition in the matrix, which was accompanied by an increased pool of deoxycholate-insoluble fibronectin. Matrix deposition of LTBP-1 could also be reduced by the competitive amine substrate putrescine. Immunolocalization at the fluorescence and electron microscopic level showed that extracellular tTgase is located at the basal and apical surfaces of cells and at cell-cell contacts, and that LTBP-1 is co-distributed with cell surface tTgase, suggesting an early contribution of tTgase to the binding of LTBP-1 to matrix proteins. LTPB-1 was also found to co-localize with both intracellular and extracellular fibronectin, and increased immunoreactivity for LTBP-1 and fibronectin was found in large molecular weight polymers in the deoxycholate-insoluble matrix of fibroblasts overexpressing tTgase. We conclude that regulation of tTgase expression is important for controlling matrix storage of latent TGF-beta1 complexes and that fibronectin may be one extracellular component to which LTBP-1 is crosslinked when LTBP-1 and tTgase interact at the cell surface. (J Histochem Cytochem 47:1417-1432, 1999)     

Targeting of cytoskeletal linker proteins to focal adhesion complexes is reduced in fibroblasts adhering to laminin-1 when compared to fibronectin

Cellular interactions with the extracellular matrix determine to a large extent cell behavior, including cell migration. These interactions take place at specialized cellular structures, the focal adhesions, which have a substrate-specific morphology. To determine the molecular and functional relevance of this observation, the composition of isolated focal adhesions developed by fibroblasts adhering to fibronectin or laminin-1 was analyzed by indirect immunofluorescence and immunoblotting with or without stabilization of the structures by cross-linking. In the absence of cross-linking, integrins, talin, vinculin and, to a lower extent, paxillin remained associated with the focal adhesions formed on both substrates, indicating a tight association of these proteins with the extracellular matrix support. By contrast, alpha-actinin, FAK, and actin were apparently loosely maintained within focal adhesions and were found associated to these structures only after stabilization by cross-linking. Interestingly, although both substrates induced clustering and aggregation of all these proteins, their relative concentration, with the exception of alpha-actinin, was lower within the focal adhesions formed on laminin-1 than in those formed on fibronectin. Moreover, as assessed in migration assays, the locomotory speed of fibroblasts was higher on laminin-1 than on fibronectin. Altogether these results indicate that integrins involved in cellular interactions with fibronectin or laminin-1 trigger the formation of focal adhesion structures which differ by molecular organization, concentration in several adhesion plaque components, and function.     

Products of cultured neuroglial cells: II. The production of fibronectin by C6 glioma cells

The possibility of fibronectin production by C6 glioma cells was examined with assays which require protein synthesis. Proteins produced by C6 cells using radiolabeled amino acid precursors were tested for affinity to collagen by binding to immobilized gelatin. The predominant collagen binding protein made by C6 coelectrophoresed with fibronectin synthesized by control fibroblasts and with the larger of the two proteins in unlabeled fibronectin when applied to polyacrylamide gels with sodium dodecyl sulfate (SDS). In addition, C6 produced a larger collagen binding protein of approximately 270,000 molecular weight. Solubilities in urea solutions of the collagen-binding proteins made by C6 cells and fibroblasts were similar. Immunofluorescence showed fibronectin associated with the C6 cell monolayer, but less abundant than the fibronectin associated with fibroblasts. Results provide evidence for the production of fibronectin by the C6 glioma cell line.     

Comparative proteomic analysis of extracellular matrix proteins secreted by two types of skin fibroblasts

The hair follicle dermal papilla is composed primarily of extracellular matrix (ECM) proteins secreted by resident fibroblasts. Dermal papilla is endowed with hair morphogenic properties, yet its composition is poorly characterized. In an attempt to understand its specificity better, we compared the protein composition of ECM secreted by cultured dermal papilla fibroblasts with that of dermal fibroblasts. ECM proteins are generally large, difficult to solubilize, and abundantly post-translationally modified. We thus implemented an original protocol for analyzing them: ECM samples were enzymatically digested directly in the culture flasks and analyzed by LC-MS/MS. Sequencing of proteolytic peptides by MS/MS yielded protein identification. The relative abundance of a given protein in dermal fibroblast versus dermal papilla samples was estimated by comparing proteolytic peptide intensities detected by MS. Using this approach, several matrix proteins were found to be present at markedly different levels in each ECM type; in particular, thrombospondin 1 and fibronectin appeared to be overrepresented in the dermal papilla fibroblast ECM. MS results were supported by Western blot and immunostaining experiments. In addition, peptide intensities were processed in two ways, which proved to favor either the quantification accuracy or the information precision at the sequence level.