The Role Of Extracellular Matrix Proteins

Many cells are coated by a layer of extracellular matrix (ECM) called the basement membrane (BM).[5] The BM is composed of two layers: an internal basal lamina (BL) linked to the plasma membrane and an external, fibrillar reticular layer. Several important ECM functions include providing substrates for cell adhesion and migration and presenting key growth factors to regulate cell growth and differentiation. ECM consists of two classes of macromolecules (Table 2). First, proteoglycans are molecules that can bind, concentrate, and present marginally soluble molecules to the cell and effectively modulate growth factor distribution and activities.[6] Proteoglycans are glycosaminoglycans that are covalently linked to protein and include perlecan, agrin, HSPG, decorin, and fibromodulin. The second class is made up of fibrous proteins that include the structural proteins collagens, elastin, and fibrillin, and the adhesive proteins laminin and fibronectin.

Collagens are the most abundant fibrous proteins in the ECM. Of the 20 collagens identified, types I, II, V, and XI are the predominant ones (Table 2).[7] Type I is the most

Table 1 Select transcription factors involved in cell differentiation of domestic animalsa'b

Transcription Preadipocytes/ Chondrocytes/

factor adipocytes Myoblast/muscle cells Endothelial cells osteoblasts

CEBP

CEBP a and CEBP S increase with adipogenesis while CEBP b remains constant (P).

PPAR

PPAR g induces adipogenesis (P, O) and increases with adipogenesis. PPAR g agonist induces UCP1 expression in SV cells (Rb).

PPAR g agonists induce adipogenesis in fibroblast like cells from skeletal muscle (B).

Smad

TGF b treatment induces Smad 2 translocation in corneal myofibroblasts (Rb). Smad 3 translocation is decreased in myofibroblasts compared to fibroblasts (P).

TGF b induces Smad 1/5 and Smad 2/3 phosphorylation and BMP 6 induces Smad 5 phosphorylation in BAEC, BCEC, and BMEC. Smad 2/3 overexpression activates PDGF B promoter and may mediate TGF b response in BAEC cells.

TGF b induces PTHrP via Smad signaling to regulate the differentiation rate of chondrocytes (A). Retinoic acid increases embryonic chondrocyte differentiation via BMP 2 induced Smad (A).

Nuclear translocation of Smad 1 and 7 induces differentiation of articular chondrocytes, whereas Smad 6 inhibits differentiation (B).

Id 2 increases with muscle atrophy following weight induced muscle hypertrophy (A).

BAEC migration and tube formation is induced via SMAD activated Id 1 expression.

Ectopic BMP 2 expression in cardiac mesoderm inhibits Pax 3 expression and impairs somite formation (A). Pax 3 and Paraxis activities are upstream of MyoD in developing embryos (A).

aA, avian; B, bovine; BAEC, bovine aortic endothelial cells; BCEC, bovine corneal endothelial cells; BMEC, bovine microvascular endothelial cells; CEBP, CAAT Enhancer Binding Protein; Id, Inhibitor of Differentiation; O, ovine; P, porcine; PDGF, Platelet Derived Growth Factor; PPAR, Peroxisome Proliferator Activating Receptor; PTHrP, Parathyroid Hormone Related Protein; Rb, rabbit; Smad, Sma , and Mad related proteins; TGF; Transforming Growth Factor.

MyoD family muscle regulatory factor (MRF) transcription factors are only expressed in muscle tissue and are not included in the table but are discussed in the text.

Table 2 Developmental studies of ECM components representing porcine (P), bovine (B), and avian (A) speciesa

components1

Preadipocytes/ adipocytes

Chondrocytes/ osteoblasts

Myoblasts /muscle cells

Collagens

IM (B): types I VI identified. V and VI remodeling important for differentiation. SQ (P): no influence of types I and IV substrata but IV expressed with differentiation.

Predominance of types I6II, IX6X (A); I6 II6 X, IX (B); I6II (P) associated with differentiation. Type II influences calcification (A) and TGF ß response via integrin signaling (B). Types I and II substrata differentially influence integrin expression (P).

Types I, III, V, and VI localized in perimysium and endomysium, IV only in endomysium and no change in localization with age (B). Type I localization associated with satellite cell myogenesis (A). Types I, III, and IV expressed in embryo (A).

Laminins

Expressed with differentiation (B,P) and substrata induces morphological differentiation (P).

Early expression in embryo (A). No change in localization with age after localization in endomysium (A,B).

Fibronectin

Decreases with differentiation (B,P) and no influence of substrata (P).

Increased and then constant expression with differentiation indicate involvement in initial attachment of early osteoblasts to pericellular matrix (A).

Decreased expression and reduced binding associated with myoblast fusion (A). Localized in fetal and embryonic connective tissue (A,B).

Integrins b1 modulates response to TGF b; b 3/BSP adhesion mediated signaling influences differentiation (B). Estrogen increases b3 expression (A). ECM influences a1 and a2 differentially (P).

a subunit ratios, cytoplasmic domains, and growth factor synergy influence myogenesis (A). a5 is critical adhesion plaque component (A). a1 is laminin/type IV collagen receptor (A).

PGs and MMPs

Age and differentiation dependent changes in PG amounts, composition, and structures that include aggrecans, biglycans, and decorin (A,B). BMP 7 enhances PG synthesis (B). MMP 3 proteolysis required for differentiation with matrix mineralization (B).

Growth or differentiation dependent changes in amounts and size of HS, HSPG, DSPG, CSPG, and glycogenin (A,B). HSPG localizes around myotubes with development (A). Developmental shift from versicon to decorin PGs (A). Decorin associated with perimysial fibrillogenesis and TGF b signaling pathway (A).

aBMP, bone morphogenetic protein; BSP, bone sialoprotein; CSPG, chondroitin sulfate proteoglycan; DSPG, dermatan sulfate proteoglycan; HS, heparin sulfate; HSPG, heparin sulfate proteoglycan; IM, intramuscular; MMPs, matrix metalloproteinases; PGs, proteoglycan; SQ, subcutaneous; TGF ß, transforming growth factor beta.

bComplete ECM substrata induce morphological differentiation of preadipocytes (P), endothelial cells (A,B), and epithelial cells (B) and are used in myoblast and satellite cell studies (P).

abundant, whereas type IV collagen has a more flexible structure and forms a meshlike structure. There are 12 laminin isoforms, each of which is a heterotrimer of alpha, beta, and gamma subunits.[8] Laminin and type IV

collagen, the major components of BL, are signaling molecules that activate signal-transducing receptors in the membrane. Fibronectin and laminin are directly involved in cell sorting and cell differentiation (Fig. 1). BL

Table 3 Proteomic and genomic techniques are useful in studying the regulation of cell differentiation3

Technology

Gene identification and quantitative expression analysis

SAGE DNA array

SNP analysis

Protein identification and quantitative expression analysis

2D gels with MS ICAT

Gene cloning Protein arrays

Protein interaction determination

In vitro protein interaction analysis

Cell map/subcellular proteomics

Multipole coupling spectroscopy

Protein structure analysis

De novo structure

Post translational modification

Protein function determination

Mutagenesis

Unique 14 base pair DNA tag used to identify gene and relative abundance. Immobilized DNA sequences spotted on a slide/chip recognize and hybridize complementary DNA in sample. Relative abundance is determined using fluorescent tagged samples.

DNA sequences compared between two individuals to reveal sequence variation.

Proteins separated by molecular weight and pI. Peptide sequences identified. Proteins are labeled, digested, and analyzed using microcapillary liquid chromatography and MS.

Convert computed sequence data into molecules that are produced and characterized. Analogous to DNA arrays. Polyclonal antibodies generated, spotted, and used to identify protein targets and protein expression.

Study protein signaling pathways by identifying binding domains, ligands, and binding affinity. Determine protein interactions and pathway characterization. Biochemical fractionation of subcellular material precedes protein identification. Pathway characterization by identifying location, orientation, and movement of proteins.

Measure frequency dependent dielectric properties. Signal transduction in whole cells, direct detection of pathway function.

Experimental determination using crystallography and/or NMR spectroscopy. Predictive structures can also be determined.

MS including TOF, MALDI, PSD, and IMAC. Limited by the nature of protein modification.

Site directed alteration of gene to investigate phenotypic changes from altered protein expression.

aSNP, single nucleotide polymorphism; SAGE, serial analysis of gene expression; MS, mass spectroscopy; ICAT, isotope coded affinity tags; NMR, nuclear magnetic resonance; TOF, time of flight; MALDI, matrix assisted laser desorption/ionization; PSD, post source decay; IMAC, immobilized metal affinity chromatography.

components not only orchestrate morphogenesis directly but also regulate development by presentation of morpho-genic, mitogenic, and trophic factors.

Integrins are cell-surface receptors responsible for cell attachment to extracellular matrices and to other cells (Table 2).[9] They effectively link the ECM to the cytoskeleton and play an important role in controlling various steps in the signaling pathways that regulate processes, such as differentiation, proliferation, and cell migration. The integrin family consists of 24 receptors assembled from combinations of 18 alpha and 18 beta chains. Matrix metalloproteinases (MMPs) are responsible for ECM breakdown and remodeling associated with morphogenesis and cell differentiation. Sixteen MMPs and several tissue inhibitors of MMPs have been identified.

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