Cell type specific recognition of the reconstituted aggregates from isolated type I collagen, type IV collagen, and type V collagen

Proc. hMian Acad. Sci. (Chem. Sci.), Vol. 111, No. 1, February 1999, pp. 171-177

© Indian Academy of Sciences

Cell type specific recognition of the reconstituted

aggregates from isolated type I collagen, type IV collagen, and type V collagen

T O S H I H I K O H A Y A S H I 2., K A Z U N O R I M I Z U N O l, M O T O H I R O H I R O S E 1, KOICHI N A K A Z A T O l, EIJIRO A D A C H I 2, Y A S U T A D A I M A M U R A 1, H I R O A K I K O S U G I 1 and K I W A M U Y O S H I K A W A 1 1Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan 2Department of Anatomy and Cell Biology, Kitasato University School of Medicine, Kitasato, Sagamihara, Kanagawa 228, Japan

e-mail: cthayas @m.komaba.ecc.u-tokyo.ac.jp

Abstract. The ultrastructure of animal tissues has a graded distribution that is reminiscent of the reconstituted aggregate structures from isolated type I collagen, type V collagen, and type IV collagen aligned in a gradient manner. We can assume that mesenchymal cells with different functions are also located in a graded way in accord with the graded distribution of collagen supramolecular aggregates. It was already known that proliferation, shape, migration, gene expression, and responses to growth factors of the cultured fibroblasts were greatly affected by the type I collagen fibrils. Other differentiated cells such as endothelial cells and smooth muscle cells are surrounded by type IV collagen and/or type V collagen more closely. The reconstituted type IV collagen aggregates in a gel form were tested as culture substrates for these differentiated cells. Of particular interest was the examination of myofibroblasts that are thought responsible for the pathogenesis of fibrotic or cirrhotic tissues. It was found that the type IV collagen gel appeared to restore the behaviors of smooth muscle-related cells including the myofibroblasts into the contractile stage.

The whole cells cultured on the type IV collagen gel were totally quiescent and formed a multicellular network. The findings suggest that interrelationships in a graded way between differentiated cells and the specific collagen aggregates might be involved in the homeostasis of tissue structure and function.

Keywords. Collagen aggregates; collagen types; cell; animal tissues; biological function.

1. Introduction

Generally, the polypeptide sequences of collagenous triple-helix contain continuous and repeated glycine-X-Y triplets with abundant proline and hydroxyproline residues on X and Y positions, respectively. Limited information is available about the supramolecular structures of collagenous proteins except for type I collagen. We have succeeded in the supramolecular assembly from isolated type IV collagen and type V collagen 1-5. In order to obtain clues to biological functions of different types of collagen, the respective aggre- gates reconstituted from isolated type I collagen, type IV collagen, and type V collagen

* For correspondence

171

172 Toshihiko Hayashi et al

were used as cell culture substrates. The response of the cell behaviors was used as probe for structural characteristic of different types of collagen aggregates and their subtle changes that might not be detected by physico-chemical or biochemical detection methods.

2. Common and characteristic features of intermolecular interactions of type I, type V, and type IV collagens through triple-helical domains

Intermolecular interactions of the collagen are governed on the one hand by the characteristics of triple-helical domain of collagenous proteins 4. Intermolecular interactions increase through the exposed hydrophobic residues on surface of the triple- helical domains at elevated temperature, resulting in laterally associated aggregates.

Staggering of the lateral association of the rod-like structures eventually conforms fibrillar aggregates.

Type V collagen molecules isolated after pepsin treatment and thus being composed of essentially triple-helical region alone :formed the fibrils with essentially the same banding pattern with the type I collagen fibrils reconstituted (figure 1). Longitudinally staggered array along the triple-helical regions may be caused by similar interactions of the collagen molecules between type I collagen and type V collagen. Thus, the lateral association of type I and type V collagen which specifically interact with charged groups and polar groups results in regularly aligned fibrillar aggregates. The staggering length is about 67 nm and appears to be' the same among the fibril-forming collagen including type II collagen, type III collagen, and type XI collagen as well as type I collagen and type V collagen.

3. Thin fibrils formed from type V collagen

Lateral acceretion of collagen triple-helices is theoretically unlimited from the model of cross-sectional arrangement model of the rod-like molecules with quasi-equivalent positioning. However, repeated experiments on reconstitution of type V collagen fibrils under various conditions demonstrated that the type V collagen fibrils as compared to type I collagen fibrils have a much smaller diameter with a very narrow range of width distribution. There must be differences in structural characteristic governing the diameter differences between fibrils of type V collagen and type I collagen. Side-chains are exposed to the surface of the collagenous triple-helices. We therefore speculate that high contents of sugar-attached hydroxylysine residues or/and bulky hydrophobic residues in the type V collagen helical domain might be responsible for limiting the accretion of the triple-helical domains (K Mizuno et al, submitted for publication).

The subtypes of type V collagen 'molecules, [al(V)]2 a2(V) and al(V)a2(V)a3(V), separated by chain composition-dependent affinity for heparin 6 formed banding fibrils with a thinner diameter than the length of D-periodicity (figure 2, left). The filament dia- meter of the type V collagen fibrils is on an average thinner than that of the type I collagen fibrils. The limited thickness of fibrils of the type V collagen is ascribed to the characteristic structure of the triple-helical domains. Type V collagen aggregates appear to lack branching at the molecular level or at the fibrillar level. The TH domain of the type V collagen has high contents of sugar-attached hydroxylysine and bulky hydropho- bic amino acid residues. These levels are much higher than those for type I collagen (table 1). The sugar-attached hydroxylysine residues and/or hydrophobic amino acid residues

Specific recognition of collagen aggretates 173

Figure 1. Surface structures of the triple-helical domain of a portion of type V collagen triple-helix. Upper helix has no sugars attached to hydroxytysine; lower helix contains two glucosyl-galactose residues attached to the hydroxylysine residues.

Table 1. Summary of the common and characteristic features of the TH (triple-helical) domains of type I collagen, type IV collagen and type V collagen in the intermolecular interactions.

Common features: Lateral associations are strong at high temperatures, hydration is high at low temperatures

Type I collagen Type IV collagen Type V collagen Interruptions of G - X - Y

Content of hydroxylysine sugar Content of bulky

hydrophobic residues Aggregate structure Thickness of filaments Gel or not

None --20 None

Little Highest High

Low High High

Fibrillar Polygonal meshwork Fibrillar 80-150 nm Less than 10 nm Less than 50 nm Usually in Gel formed under Gel is not formed gel form limited conditions

may limit the growth of the lateral association of triple-helices. The intermolecular interactions of the triple-helical domains depend greatly on the surface structure of the triple-helical domains (figure 1). Virtually all amino acid side chains except the Gly residues in the collagenous domains are exposed to the surface. Hydrophobic side chains prefer homologous residues in the interactions, while those with charged groups or polar groups interact in a complementary way (K Mizuno et al, submitted for publication).

! 74 Toshihiko Hayashi et al

Type IV meshwork Type V fibrils Type ! fibrils

1 0 0 n m 1 0 0 0 n m 1 0 0 0 n m Figure 2. Aggregate structures of type IV, type V and type I collagens reconstituted from the isolated proteins.

4. Polygonal meshwork gel reconstituted from the type IV collagen

We showed that bovine lens capsule type IV collagen formed a gel under rather unique conditions 1.2, as compared to the conditions required for the type I collagen gel formation. The type IV collagen gel comprises polygonal meshwork with a pore size of about 18 nm 3, resembling in dimension the basal lamina skeletal meshwork (figure 2, left). A fine meshwork of type IV collagen aggregate seems to originate from branching at the molecular level, which could be the triple-helical interruption sites. The intermolecular interaction of the type IV collagen resulting in branching or crosslinking of the filaments is ascribed to the NC 1 (non-triple helical domains) domain that binds to the type IV collagen triple-helical domain (K Nakazato et al, manuscripts in preparation).

NC1 domain-free type IV collagen obtained by pepsin ot chymotrypsin treatment did not form gel. Either pepsin-treated type IV collagen comprising helical-domains or bacterial collagenase-treated type IV collagen comprising NC1 domains, inhibited the gel formation of type IV collagen which was extracted from bovine lens capsules with acetic acid (K Nakazato et al, manuscripts in preparation). However, since gly-X-Y repeats of the type IV collagen triple helical regions are interrupted, the overall shape of the triple helix might be kinked at the interrupted sites. Thus the lateral association of the type IV collagen triple-helical regions might branch off one another at such kinked sites 4. In fact, we observed polygonal meshwork structures for the aggregates reconstituted from intact type IV collagen extracted from lens capsule without proteolytic treatment 2. The meshwork with an average pore size of 18 nm was seen when distances between branching points were measured. We are thus in favor of the idea that the helical regions are responsible for the determination of meshwork pore size. Pepsin-treated type IV collagen with sufficiently long lengths of the helical regions would form meshwork structure under the conditions where lateral association is allowed. In fact, we observed polygonal meshwork formation from the pepsin-treated placenta type IV collagen 3.

Specific recognition of collagen aggretates 175

Type IV collagen-coated plate Type I collagen gel

Type IV collagen gel

Figure

3. Morphology of rat hepatic stellate cells on different substrates.

5. Cellular recognition of type I collagen gel

Fibroblasts cultured in type I collagen gel showed repressed growth, repressed production of collagenous proteins, and repressed response to various growth factors or cytokines.

Elongated morphology was assumed by the fibroblasts cultured within type I collagen

gel7-9.

This phenomena could be explained by the fibroblasts recognition of the type I collagen aggregates as appropriate environment. If the fibroblasts recognize the environments as insufficiently organized, they would start to either produce or rearrange the type I collagen fibrils. As shown in table 2, on type IV collagen gel consisting of polygonal meshwork, fibroblasts proliferated and showed morphology similar to the fibroblasts on type I collagen coated dish or on the plastic. Type IV collagen gel distinguished fibroblasts from myofibroblastic cells, though these cells have many features in common including the responses to type I collagen environments.

6. Cell culture on type IV collagen gel

The stellate cells change the shape, proliferate, and produce fibrillar collagens, when the cells are cultured on plastic dish, type I collagen, type I collagen gel,' and on Matrigel. On the other hand, the ceils showed a unique morphology with an extremely elongated shape when cultured on the type IV collagen gel whose terminals could not be identified and appeared to be connected with the processes of adjacent cells (figure 3 ) 4 . T h e hepatic stellate cells, little proliferated on the type IV collagen gel, while on other substrates type I collagen gel, type I collagen molecules, type IV collagen molecules, and plastic dish, the

176 Toshihiko Hayashi et al

6

N

z

1

Figure 4.

~, Type IV

collagen-coated

4 ....~ Type I collagen-coated

Plastic

J J y

3 ~

2

1 ~ ~ Type IV

^collagen

I

I I

^{I r}

2 4 6 8 10 12

Culture days

Growth curves of primary cultures of stellate cells.

Table 2. Cell shape and growth cultured on collagen aggregates.

Cultured cell type Type I collagen Type IV collagen

Fibroblasts Cell shape Bipolar Bipolar

Cell growth Repressed Accelerated Smooth muscle cells, Cell shape Bipolar Cell processes and

hepatic stellate cells, junction formation,

and mesangial cells Cell growth Not affected repressed Rhabdomyosarcoma Cell shape Bipolar, Cell processes and

junction formation Cell growth Not affected repressed

cell grew with a similar growth rate (figure 4). Since the original cell number used was adjusted to be the same, the data in figure 4 suggested several intriguing points. On day 2 cell number on type I collagen gel was as low as cell number on type IV collagen gel where no significant growth was observed later. Cells were delayed in starting proliferation on type I collagen gel, The plate coated with type IV collagen molecules appeared to provide the best substrate in terms of cell proliferation. Of particular interest was that the type IV collagen had the strongest influence on hepatic stellate cell proliferation, either positively or negatively. They apparently switch on and off the proliferation upon changing the protein aggregation states. It might imply that the type IV collagen protein contains a regulatory site(s) for cell growth that may work in a concerted manner.

Specific recognition of collagen aggretates 177 If we look at this phenomenon from a different side, the hepatic stellate cells are very good probes for the aggregate structure of type IV collagen. In fact, vascular smooth muscle cells and kidney mesangium cells, that are said to have similar positions to vascular endothelial cells and presumably similar function such as contractility, showed exactly the same phenotypes to the type IV collagen substrates, while fibroblasts that are otherwise similar to these cells showed distinct behavior. Fibroblasts does not recognize type IV collagen gel and grew on the type IV collagen gel as rapidly as on the plastic.

Acknowledgements

The present study was supported in part by the Scientific Research Grant from the Ministry of Education, Culture, Sports and Sciences of Japan, Grant-in-Aid for Scientific Research on Priority Areas (09229219 Functionally Graded Materials, 09217210 Supramolecular Structure), Grant-in-Aid for Developmental Scientific Research (07558249), by the Japan Society for the Promotion of Science, "Research for the Future"

Program (JSPS-RFTF96100201) and by the Program for Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research

(OPSR).

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