DEVELOPMENT, TISSUE REMODELING AND DISEASE

John Callahan, Bob Dylan, Eric Clapton or David Bowie? Where in the world is Pori? What Lewis Wolpert has to do with origami? Save the electrons!

What is ECM? Growth and development critically depend on tissue remodeling and the exquisite balance between making and breaking the extracellular matrix (ECM) of cells and tissues. The ECM is vital for cells to do what they are supposed to do during growth and development. Divide, differentiate or die. The ECM is part of the cell. Where does a cell begin and end? (Fig.1).

The ECM is made of glycoproteins, collagens, proteoglycans and glycosaminoglycans, and its structural details and function differ between different tissues and organs. More than 80 genes code for different ECM components including 34 genes for the collagen family of proteins, which are the most abundant proteins constituting 30% of total body proteins.The ECM is particularly abundant in connective tissues such as bones, ligaments, tendons and cartilage.

The ECM has many functions. It is the glue that supports and keeps tissues and organs together. It regulates cell behavior and can tell cells what to do. It can play a permissive or instructive and morphogenetic role in growth and development. It can be good, bad and ugly. This is most dramatically illustrated by a variety of devastating genetic diseases and developmental disorders affecting the skeleton, blood vessels, skin and many other organ systems. A large number of such diseases are caused by gene mutations for collagens and other ECM components. How can this happen, how can genetically altered mutant ECM components cause these diseases? Is there anything common in the molecular pathogenesis of these diseases? What can be done to treat patients suffering from these diseases? What is the function of the ECM in normal development and health anyway?

What is ECM signaling?No cell is an island. Cells interact and communicate with other cells and with the ECM. Cells need signals to divide, differentiate and die or not to die. In the end it all depends on the regulation of gene expression and the activity of different gene products in different cells. How does the ECM influence genes? That's ECM signaling.

What are MMPs? Proteolytic degradation and remodeling of the ECM is largely controlled by a superfamily of Zn(2+)-dependent extracellular enzymes called matrix metalloproteinases (MMPs) and their specific tissue inhibitors of metalloproteinases or TIMPs. To date, 15 genetically distinct MMPs have been cloned and characterized from humans, in addition to four novel MMPs from other species. MMPs share similar domain structure, have different but often overlapping substrate specificities and are grouped as collagenases, gelatinases, stromelysins and membrane-type MMPs (MT-MMPs)(Fig.2).

Collagenases (MMP1, MMP8 and MMP13) are the only enzymes that can cleave fibrillar collagens such as collagen types I, II, III, VII and X.

Gelatinases A and B, also known as 72 kDa and 92 kDa type IV collagenases (MMP2 and MMP9), digest denatured collagen (gelatin) and other ECM components including basement membranes, a special type of ECM of endothelial, epithelial, fat, muscle and peripheral nerve cells.

Stromelysins (MMP3, MMP10 and MMP11) are wide spectrum enzymes, and stromelysin-1 (MMP3) degrades almost any ECM component including cartilage proteoglycans. Also, stromelysin-1 proteolytically activates other MMPs such as interstitial collagenase (MMP1), matrilysin (MMP7), collagenase-2 (MMP8), 92 kDa type IV collagenase (MMP9) and collagenase-3 (MMP13) suggesting that stromelysin-1 plays a special "upstream" role in ECM degradation and tissue remodeling.

MT-MMPs represent a recently discovered novel class of MMPs. Unlike other MMPs, they are not secreted but remain anchored to the cell surface via their carboxyl-terminal transmembrane domain. At present, four different MT-MMPs have been cloned and characterized. MT-MMPs digest ECM components and proteolytically activate other MMPs such as 72 kDa type IV collagenase and therefore, may regulate pericellular matrix degradation at the cell surface.

Many MMPs are expressed widely during embryogenesis, but not in adult life, often in a highly cell and tissue-specific manner suggesting distinct roles for different MMPs in growth, development and tissue remodeling. In adult life, MMPs are expressed in rapidly remodeling tissues such as the term placenta, menstrual endometrium, involuting mammary glands, and during wound healing and inflammation.

MMPs are also thought to play a critical role in tumor growth and metastasis, and in the progression of other diseases such as arthritis, atherosclerosis and aneurysm.

MMP structure and function. MMPs have four major domains with distinct structure and function. An N-terminal propeptide (PP) or inhibitory domain of about 80 amino acids, which is proteolytically removed from the active MMPs. This domain contains an evolutionary conserved sequence PRCG-PD in which the free Cys-residue is critical for keeping MMPs inactive.

A catalytic domain (CD) of about 180 amino acids contains a highly conserved sequence HE-GH-LGL-H that provides three binding sites (His-residues) for the catalytic metal ion Zn(2+). The Cys-residue in the propeptide of inactive MMPs provides the fourth binding site for Zn(2+). In activated MMPs, this site is taken up by a water molecule, which is also hydrogen bonded to the conserved glutamic acid (E). This presumably destabilizes and "activates" the bound water molecule, a process that facilitates hydrolysis of a peptide bond of the target substrate.

Another conserved sequence DD--GIQ has a catalytic Asp-residue. Mutation of this aspartic acid (underlined) to glycine in neutrophil collagenase-2 (MMP8) completely abolished its proteolytic activity on casein and native fibrillar collagen. This aspartyl-residue probably has a conformation stabilizing role and, in the crystal structure of collagenase-2, it forms hydrogen bonds to the so-called Met-turn residues Leu214 and Met215. Met-turn is a characteristic sequence Ala-Leu-Met-Tyr (with an invariant methionine) of zinc-dependent metalloproteinases including MMPs. The catalytic domain is a compact structure made of three alpha-helices and a twisted five-stranded beta-sheet.

A linker peptide (LP) of 17-72 amino acids, an unstructured random coil, links the catalytic domain to a C-terminal domain of MMPs. LP is rich in proline and basic amino acids (Arg, Lys), and its only conserved feature is that it begins with glycine and ends with proline. LP, which is the most variable domain, is involved in the substrate selection of MMPs.

The C-terminal domain (VD) of about 200 amino acids is composed of four tandem repeats of a motif found in vitronectin and pexin-family of proteins. The C-terminal domain is a compact structure that looks like a four-bladed propeller. Each blade of the MMP propeller is made of an antiparallel four-stranded beta-sheet and the adjacent blades are arranged perpendicularly around a funnel-shaped tunnel. The C-terminal domain is important for protein-protein interactions, it binds to TIMPs and is involved in the substrate selection of MMPs.

Several MMPs have extra domains. Gelatinase A and gelatinase B (MMP2 and MMP9) contain three tandem repeats (each encoded by a separate exon) of a motif found in fibronectin that form a 176-amino acid-long fibronectin-like domain (FD). MT-MMPs contain a C-terminal transmembrane (TM) domain of 24 amino acids followed by a cytoplasmic tail domain (TD) of 20 amino acids.

Some MMPs lack domains. Matrilysin (MMP7) lacks the linker peptide and C-terminal vitronectin-like domain altogether, and XMMP, a novel MMP from Xenopus laevis, has no linker peptide.

MMPs are extremely slow enzymes. One stromelysin-1 (MMP3) molecule cuts one substrate molecule in 8 minutes. In comparison, carbonic anhydraze, a Zn(2+)-dependent metalloenzyme and the fastest enzyme there is, speeds up the reaction H2O + CO2 = HCO3(-) + H(+) 10 million-fold. It catalyzes the reaction 100,000 times per second.

MMP regulation

What are TIMPs?MMPs are inhibited by naturally occuring proteins called tissue inhibitors of metalloproteinases (TIMPs). Four genetically distinct TIMPs have been cloned and characterized in humans. Of interest is that TIMPs also have mitogenic and cell growth promoting activity that is separate from their MMP inhibition activity. Also, TIMP3 is a component of the ECM and constituent of some basement membranes.

TIMP structure and function

TIMP regulation

Why are there so many MMPs? At present, 15 different MMPs have been characterized in humans. However, recent cloning of four novel MMPs from other species suggests that there are at least 19 different MMPs. Why are there so many MMPs? Although the substrate selection of MMPs is large (and not restricted to ECM components only), 19 different MMPs with overlapping substrate specificities and similar kinetics seems redundant. Is it because MMPs often are, for some unknown reasons, highly cell and tissue-specific. For example, a novel MMP called enamelysin is expressed in the tooth's enamel organ, and collagenase-2 (MMP8) is expressed only in neutrophils and cartilage articular chondrocytes. Or is it because ECM degradation and remodeling by MMPs is involved in cell and tissue interactions that in turn specify cell fate and regulate the development of the embryo. Consider that in health as well as in disease, the ECM is a rich source of signals. Many of the signals can be "tailor-made" by specific cleavage of ECM components by different MMPs to fit the individual needs of different cell types.

ECM and MMP signaling in development, tissue remodeling and disease.Compared to cell signaling by secreted and soluble factors, such as growth factors and cytokines, little is known about cell signaling by the extracellular matrix (ECM) and how it specifies cell fate and regulates the formation of tissues and organs during early development. The ECM is a ubiquitous and dynamic structure of collagens, glycoproteins and proteoglycans, and its degradation and remodeling is largely controlled by proteolytic enzymes called matrix metalloproteinases (MMPs) and their specific naturally occuring tissue inhibitors of metalloproteinases (TIMPs).

MMPs and TIMPs in disease

Arthritis

Cancer

Cardiovascular diseases

Sorsby's fundus dystrophy

Mutations in the TIMP3 gene cause a rare eye disease called Sorsby's fundus dystrophy (SFD). SFD is characterized by loss of central vision associated with subretinal neovascularization and atrophy of the choriocapillaris (CL), retinal pigment epithelium (PE) and retina. A hallmark of SFD is thickening of the Bruch's basement membrane (BM) between the PE and choroid. This may lead to visual complications of SFD by stimulating new blood vessel growth from the choroid through Bruch's membrane into the subretinal space. Remarkably, SFD is a dominant disease with onset of symptoms in the third and fourth decade, and can be effectively treated with high doses (50,000 IU/day) of orally taken vitamin A.

Markku Kurkinen

Ning Chen

Maozhou Yang

August 23, 1997