[12] Modulation of elastin synthesis: In vitro models

232

MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

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Degradation. The contribution of extracellular degradation to final production values appears to be minimal in most cell cultures. Intracellular degradation of elastin peptides is difficult to measure, as the peptide represents a relatively small percentage of the total protein produced and no specific peptide marker is available. Tropoelastin is much more susceptible to proteolysis than insoluble cross-linked elastin. Tropoelastin secreted by human skin fibroblasts in ,culture is rapidly fragmented to approximately five peptides, which then appear to be stable for at least 60 hr, as shown by pulse-chase studies (Fig. 4). This fragmentation pattern is reminiscent of that seen for tropoelastin purified from aortas of copper-deficient swine or lathyritic chicks, where a serine proteinase activity probably is responsible for cleavage. The intact peptide fragments of the protein do not appear to undergo complete degradation, however. Proteolysis is measured by adding HPLC-purified 125I-labeled tropoelastin (a single band on SDS-PAGE), which has been iodinated using Iodogen (Pierce), to cultures and assaying counts recovered in the TCA-insoluble pellet over time. 34,35 Tropoelastin degradation in the culture medium of normal human skin fibroblasts over a period of 72 hr is negligible using this assay procedure. Tropoelastin fragments present in the medium of cultured cells are recognized by the rabbit anti(pig)-o~elastin antibody. Radiolabeled ~25I-labeled tropoelastin peptides (intact and fragmented) are detected by immunoblotting an 8% SDS-polyacrylamide gel of immunoprecipitated peptides and comparing the banding pattern of the Western blot (to detect immunoreactive fragments) to an autoradiogram of the same blot (to detect radiolabeled fragments) (Fig. 3).

Acknowledgments We thank Gwenevere

Shaw for preparation of the manuscript.

[12] M o d u l a t i o n of E l a s t i n S y n t h e s i s : I n Vitro M o d e l s

By ROBERT P. MECHAM The unique physical properties of elastin have hindered the elucidation of factors or pathways involved in regulation of elastin accumulation. Most of our knowledge of modulation of elastin synthesis is therefore derived from in vitro studies where culture conditions can be closely METHODS IN ENZYMOLOGY, VOL. 144

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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controlled and where cross-linking of elastin precursor molecules can be effectively inhibited. While studies of this kind have provided important insights into regulatory pathways in cultured cells, it should be stressed that the elastin phenotype is extremely unstable in vitro and that conclusions from manipulation of cultured cells may not be relevant for cells in situ until direct comparison of both conditions is possible. Nevertheless, cell culture techniques remain one of the most useful tools for studying elastin metabolism. In this chapter we will first examine methodology for maintaining elastin-producing cells in culture, and then consider factors that modulate elastin production. Techniques for Culturing Elastin-Producing Cells Numerous cell types synthesize elastin in vitro, including aortic smooth muscle cellsf1,2 dermal fibroblasts, 3 fibroblasts from ligamentum nuchae, 4 ear chondroblasts, 5,6 lung mesothelial ceUs,7 bovine corneal endothelial cells, 8 and vascular endothelial cells. 9,r° All of these cell types express the elastin phenotype when maintained under appropriate culture conditions although it should be emphasized that how one assays for elastin is an important parameter in assessing levels of elastin synthesis (see the chapter by Wrenn and Mecham [13] in this volume). Ligamentum Nuchae Fibroblasts

Fibroblasts from bovine or sheep ligamentum nuchae are established using the explant technique. Ligament tissue is quickly removed from the animal using sterile instruments and placed immediately into an ice-cold R. Ross, J. Cell Biol. 50, 172 (1971). z B. Faris, L. L. Salcedo, V. Cook, L. Johnson, J. A. Foster, and C. Franzblau, Biochem. Biophys. Res. Commun. 418, 93 (1976). 3 M. G. Giro, A. I. Oikarinen, H. Oikarinen, G, Sephel, J. Uitto, and J. M. Davidson, J. Clin. Invest. 75, 672 (1985). 4 R. P. Mecham, G. Lange, J. Madaras, and B. Starcher, J. Cell Biol. 90, 332 (1981). 5 j, M. Field, G. W. Rodger, J. C. Hunter, A. Serafini-Fracassini, and M, Spina, Arch. Biochem. Biophys. 191, 705 (1978). 6 G. Quintarelli, B. C. Starcher, A. Vocaturo, F. Di Gianfilippo, L. Gotte, and R. P. Mecham, Connect. Tissue Res. 7, 1 (1979). 7 S. I. Rennard, J. C. Jaurand, J. Bignon, O. Kawanam, V. J. Ferrans, J. Davidson, and R. G. Crystal, Am. Rev. Respir. Dis. 130, 267 (1984). D. K. Fujii and R. P. Mecham, manuscript in preparation. 9 W. H. Carries, P. A. Abraham, and V. Buonassisi, Biochem. Biophys. Res. Commun. 90, 1393 (1979). 10 R. P. Mecham, J. Madaras, J. A. McDonald, and U. Ryan, J. Cell. Physiol. 116, 282 (1983).

234

MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

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sterile physiological buffer supplemented with antibiotics. The tissue should be kept on ice during transport to the laboratory. Under sterile conditions, the tissue is cleaned of adherent muscle and fascia and then minced into small (approximately 1 mm 3) pieces with a sterile scalpel. The pieces are suspended in culture medium, taken up in a sterile 10-ml pipet, and dispensed into T-75 culture flasks that contain 3 ml of culture medium. The pieces (30-60 per flask) are dispersed over the bottom of the flasks by swirling and the flasks are tilted gently upright. Medium with unattached explants is removed and added to other T-75 flasks. This process is repeated until all of the explants are attached. Ten milliliters of growth medium is then added and each flask is left on its side in the incubator for 1-2 hr so that explants attach firmly to the plastic surface. The flasks are then layed gently back down, allowing growth media to cover the explant pieces. Explant cultures are left undisturbed until cell growth is well established. Outgrowth of cells should begin within 5-10 days, depending upon the age of the tissue. Cells from a late gestation bovine fetus, for example, should be ready to subculture 2-3 weeks after explanting. Because elastin production by ligament cells is under developmental regulation, it is important to be aware of the developmental age of the ligament tissue. Elastin production is highest during the last developmental trimester and in the early neonatal period H but decreases to low levels in the adult. Cells from fetuses younger than 120 days make little or no elastin. Despite a high level of elastin synthesis, ligament cells do not crosslink soluble elastin monomers to form insoluble elastin. This feature of in vitro culture with this particular cell type has proven to be of great advantage for studying modulation of elastin production since secreted tropoelastin remains soluble and can be quantified easily by immunoassay techniques. Smooth Muscle Cells

Techniques for establishing cultures of vascular smooth muscle cells from explants of aortic medial tissue have been described in detail in a previous volume of this series ~2and are similar to those described above for ligament fibroblasts. It is important to select aortic tissue during the late gestational or early neonatal period when elastin production is most pronounced. It is also important to note that all aortic smooth muscle cells

n R. P. Mecham, S. L. Morris, B. D. Levy, and D. S. Wrenn, J. Biol. Chem. 259, 12414 (1984). 12 C. Franzblau and B. Faris, this series, Vol. 82, p. 617.

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235

may not be phenotypically equivalent. Recent studies have shown that elastin production by cells in the thoracic aorta is substantially greater than rates of production in areas of the aorta further removed from the heart.13 In studies of elastin production by vascular smooth muscle cells it is therefore important to consider developmental history as well as the derivation of these cells. Ear Chondroblasts

Chondroblasts can be harvested directly from ear cartilage after collagenase digestion. Cells from rabbits and cows have been cultured successfully using this technique. The skin of the ear is stripped from the cartilage under sterile conditions and any adherent material is removed by scraping with a scapel. The cleaned cartilage is chopped into small pieces and incubated at 37° with constant stirring in filter-sterilized 50 mM HEPES buffer, pH. 7.4, containing 0.01 M calcium acetate, 150 mM NaCI, antibiotics, and 2 mg/ml collagenase (Sigma, type I). When the tissue has nearly dissolved (2-3 hr, depending on the maturity of the cartilage) the digest is transferred to a sterile conical tube and swirled to suspend cells and undigested debris. Any undigested cartilage fragments that settle quickly to the bottom are removed by aspiration with a sterile pipet or by filtration through sterile gauze. The cells are pelleted by centrifugation at 500 g for 10 min. Collagenase buffer is decanted from the cell pellet and the cells are resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. The cells are again pelleted by centrifugation, resuspended in DMEM-10% fetal bovine serum, and are plated into tissue culture dishes. The onset of elastin production in developing ear cartilage occurs much earlier than in aorta or ligament tissue. Our studies have shown that bovine ear chondroblasts produce elastin as early as cartilage can be recognized morphologically in the developing ear bud. We prefer to use ear cartilage from mid-gestation bovine fetuses (around 140-160 days) because it is difficult to separate the skin from the cartilage in fetuses much older than 180 days. This does not seem to be as difficult a problem with ear tissue from neonatal rabbits, however. Dermal Fibroblasts

Techniques for culturing fibroblasts from dermal explants are similar to those described for smooth muscle cells and ligament fibroblasts. During the developmental period elastin production in the skin is highest 13 j. M. Davidson, K. E. Hill, M. L. Mason, and M. G. Giro, J. Biol. Chem. 260, 1901 (1985).

236

MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

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during the later stages of gestation. Little information is available on rates of elastin synthesis in aging skin, however. Although elastin production by dermal fibroblasts is relatively low the elastin phenotype shows greater stability in dermal fibroblasts in culture than most other cell types) Lung Pleural Mesothelial Cells and Bovine Corneal Endothelial Cells Bovine corneal endothelial cells and cells from the parietal pleura of the rat represent two elastin-producing cell types that organize as a single cell layer resting on a basal lamina. In tissue culture, elastin is found beneath each cell type in an extensive extracellular matrix that resembles basal lamina. 7,8 Rat pleural mesothelial cells are isolated by superficial, gentle but firm scraping of the parietal pleura with a Desmarres scarificator.~4 The cells are cultured in medium containing 10% fetal bovine serum and are subcultured with trypsin. Explants of cells from superficial scrapings begin to form colonies within 24-48 hr. The cells have a characteristic, purely epithelial appearance and form a monolayer of polygonal cells in intimate contact with each other. Cells from the submesothelium, however, resemble smooth muscle cells in appearance. If scraping of the parietal pleura is too deep, mixed primary cultures of both cell types are obtained. The corneal endothelium is a neural crest-derived, simple squamous epithelium that covers the posterior surface of the cornea. Corneas are removed from adult bovine eyes and the endothelial cells are dislodged from Descemet's membrane by incubating the endothelial surface at 37° for 5-6 min with a balanced salt solution buffered to pH 7.3 with 15 mM HEPES containing 5 mM EDTA and 0.18% crude trypsin (1:250). Retracted cells are dislodged using a silicone rubber spatula taking care to avoid cutting the stromal surface. The dislodged cells floating in the trypsin-EDTA solution are aspirated from the eye, washed with medium contain 10% fetal bovine serum, and subsequently plated into tissue culture dishes. Interestingly, the inclusion in culture medium of 5% dextran (Mr 40,000) increases both the amount of extracellular matrix (ECM) that is organized beneath the celP 5 and the amount of elastin associated with that matrix: Rat Aortic Smooth Muscle Cells Rat aortic smooth muscle cells are efficient producers of elastin in culture. Aortae from 1- to 4-day-old rats are cut into 1-mm 2 pieces, trans14 M. C. Jaurand, J. F. Bernaudin, A. Renier, H. Kaplan, and J. Bignon, In Vitro 17, 98 (1981). 15 D. Gospodarowicz and C. R. Ill, Proc. Natl. Acad. Sci. U.S.A. 77, 2726 (1980).

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237

ferred to a sterile 10-ml capped centrifuge tube, and digested at neutral pH in a physiological buffer containing a mixture of collagenase and elastase (1 mg of Sigma type I collagenase and 0.25 mg of Sigma type II chromatographically pure elastase per ml of buffer). Digestion is allowed to proceed until approximately 80% of the cells have gone into suspension (about 1 hr). Cells are washed twice with medium, evaluated for viability using trypan blue, and plated in medium containing 20% fetal bovine serum, 16 Matrix-Free Chick Embryo Aorta Cells Aortas and associated large blood vessels are dissected from 17-dayold chick embryos and matrix-free aorta cells are isolated from tissues by limited proteolytic digestion. 17 The large blood vessels from 10 chick embryos (about 300 mg wet weight) are incubated in 1 ml of medium containing 1.3 mg collagenase (Sigma type I) and 2.5 mg of trypsin for 90120 rain. Digestion is stopped as soon as it becomes apparent that most of the tissue is dispersed. The digest is filtered through sterile gauze and cells are recovered by centrifugation at 600 g for 6 min. The cells are washed by centrifugation two more times in culture medium containing 10% fetal calf serum and are resuspended in culture medium containing 10% fetal bovine serum at a concentration of 5-7 million cell/ml. Incubations are carried out with moderate shaking at 37° . Effects of Culture Conditions on Elastin Production Cell Density, Cell Passage, and Fetal Bovine Serum The elastin phenotype is extremely unstable in vitro and care must be taken to adjust culture conditions to optimize for elastin production. Cell density and passage number are variables that alter significantly the rate of elastin synthesis. For example, tropoelastin synthesis per cell by fetal bovine ligament fibroblasts 4 and smooth muscle cells 3 is low during logarithmic growth and highest at the late log phase. After reaching confluency elastin production decreases as cultures become more dense. Likewise, elastin synthesis decreases rapidly with either serial subculture or with age in culture. After four trypsinizations over a period of 6 weeks, for example, soluble elastin production by ligament fibroblasts decreases to only 10-20% of the level observed with first passage cells. Soluble

16 B. W. Oakes, A. C. Batty, C. J. Handley, and L. B. Sandberg, Eur. J. Cell Biol. 27, 34 (1982). T7j. Uitto, H. P. Hoffman, and D. J. Prockop, Arch. Biochem. Biophys. 173, 187 (1976).

238

MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

[12]

elastin production by sixth-passage cells is much less sensitive to dexamethasone stimulation than is elastin production by first passage cells. 4 The addition of fetal bovine serum to cultured cells increases elastin production manyfold. Figure 1 shows that maximal elastin production by FCL fibroblasts is obtained with serum concentrations equal to or greater than 5% (v/v). Calf serum or medium containing serum substitutes is not as effective as fetal bovine serum in stimulating elastin synthesis. While serum levels greater than 5% may prove optimal for cell growth, serum components can interfere with immunoassay techniques for quantifying elastin production. High immunoassay background values can be reduced appreciably, however, when fetal calf serum is preabsorbed with antielastin IgG bound to Sepharose. It is sometimes necessary to maintain cells in high serum concentrations during the growth period but to use lower serum levels in medium that will be collected for analysis. Even so, a lower serum level should be selected that still provides adequate stimulation of elastin production. It is important to be aware that variability between batches of fetal bovine serum can have marked effects on elastin production by cultured cells. It is advisable to test numerous serum lots for stimulation of elastin production as well as for maintenance of cell growth. A large volume of the most effective lot can then be purchased to assure reproducible conditions between experiments. Table I compares the composition of numerous serum components in newborn calf serum and five different lots of 16FA [ ] Without ~APN 14"

•

50 pg/ml ~APN

5.6

T i

~, ~ 10

~'~

5.2

~

4.8

"

4.4

8

~

O~6

u

m~ 4 c0

0

0

2

4.0 3.6

0

2.5 5.0 7.5 10.0 0 2.5 5.0 7.5 10.0 % FETAL CALF SERUM IN CULTURE MEDIUM FIG. 1. Effects of fetal bovine serum on elastin (A) and total protein (B) production by fetal calf ligamentum (FCL) nuchae fibroblasts. Elastic production was determined by radioimmunoassay and total protein synthesis by trichloroacetic acid precipitation of [3H]valine-labeled protein. The apparent decrease in elastin production at serum levels greater than 5% is an artifact due to interference of serum components in competitive binding radioimmunoassays.

[12]

239

MODULATION OF ELASTIN PRODUCTION

TABLE I BIOCHEMICAL ANALYSIS OF FIVE DIFFERENT LOTS OF FETAL BOVINE SERUM AND NEWBORN CALF SERUMa

Fetal calf serum lot number

Triglycerides (mg/dl) Cholesterol (rag%) Phospholipids (mg/dl) LDL (mg/dl) HDL (mg/dl) Free fatty acids (mEq/liter) Cortisol (/xg/dl) Progesterone (ng/dl) Testosterone (ng/dl) Corticosterone (ng/dl) Growth hormone (ng/ml) Insulin (/xU/ml) Thyroxine T4 (/zg/dl) PTH (pg/ml) /3-Estradiol (pg/ml) PG E, A, B (mg/ml) PGF (ng/ml) PGE2 (pg/ml) PGF2~ (pg/ml)

346

401 b

413

438

506 b

32 25 -16 12 -<0.6 <1 34 35 33.3 5 11.9 2439 14.3 6.5 6.7 ---

<1 51 63 -9 0.03 0.3 I1 -47 48.1 9 11.8 2110 7 16.5 6 310 2600

11 38 123 16 20 <0.01 0.5 6 8 35 66.3 3 13 -1.4 17.02 4.9 350 2700

20 35 -24 7 <0. I 0,3 14 11 39 -4 12.8 2358 ------

1 42 55 -<10 -0.4 11.2 6.4 --4.4 14.3 -9 -----

Newborn calf serum 12 -117 48 71 0.3 2.8 49 63 326 15 12 3236 ------

" Analyses provided by Hyclone Laboratories, Logan, Utah. b Serum lots that produced maximal elastin production by ligamentum nuchae fibroblasts.

fetal bovine serum. Low levels of elastin production were observed with c a l f s e r u m a n d t h r e e o f t h e f e t a l s e r u m l o t s w h i l e e l a s t i n p r o d u c t i o n almost doubled in the other two. It has been our experience that serum lots with low triglyceride levels are best for maintaining the elastin phenotype of ligament cells, although this conclusion may not be true for other cell types.

Insoluble Elastin Formation N o t all c e l l t y p e s a r e c a p a b l e o f f o r m i n g i n s o l u b l e e l a s t i n i n c u l t u r e . Greater than 90% of the tropoelastin secreted by ligament and dermal fibroblasts remains soluble in the medium whereas chondrocytes and rat aortic smooth muscle cells retain a majority of the secreted elastin in the cell layer as cross-linked, insoluble elastin. Bovine or rabbit aortic smooth

240

MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

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muscle cells secrete most of their elastin into the medium in early culture but as extracellular matrix accumulates with time, tropoelastin becomes associated with the cell layer so that substantial amounts of insoluble elastin begin to accumulate a few weeks after confluency. The propensity of a particular cell line to form insoluble elastin should be an important consideration when designing experiments. Inhibitors of cross-link formation, such as fl-aminopropionitrile (/3APN), should be included during all incubations in order to assure quantitative recovery of soluble elastin.

Ascorbic Acid Ascorbic acid is an important cofactor for hydroxylation of proline residues and cultured cells grown in the presence of ascorbate accumulate more collagen than cells grown without ascorbic acid. The effects of ascorbate on elastin production, however, are varied and act to alter accumulation of insoluble elastin more than changing synthesis of the soluble elastin precursor. Therefore, cells that do not elaborate an extensive extracellular matrix are less affected by ascorbate addition than cells that accumulate an abundant pericellular matrix. Ligament fibroblasts in culture synthesize high levels of tropoelastin but form little cross-linked elastin. The inclusion of up to 50/zg/ml ascorbate in cell culture medium does not alter total tropoelastin synthesis 4 when compared with scorbutic cultures. In contrast to the sparse matrix of the ligament fibroblast, smooth muscle ceils accumulate a more abundant ECM that includes both collagen and insoluble elastin. In the presence of 0.5 t~g/ml ascorbate, insoluble elastin accumulation is unaffected in rabbit aortic smooth muscle cell cultures. However, when the ascorbate concentration is raised to 2/xg/ml or higher, insoluble elastin accumulation decreases. 18 The effect is even more dramatic with rat aortic smooth muscle cells. In the absence of ascorbate, rat cells accumulate an insoluble elastin component which can account for as much as 50% of the total protein in the extracellular matrix. In the presence of ascorbate, the amount of insoluble collagen increases while insoluble elastin decreases significantly, perhaps as a result of overhydroxylation of tropoelastin.19 By altering the ascorbate conditions of rat aortic cells, extracellular matrices with varying ratios of elastin to collagen can be obtained.

~8 B. Faris, R. Ferrera, P. Toselli, J. Nambu, W. A. Gonnerman, and C. Franzblau, Biochim. Biophys. Acta 797, 71 (1984). ~9L. M. Barone, B. Fads, S. D. Chipman, P. Toselli, B. W. Oakes, and C. Franzblau, Biochirn. Biophys. Acta 8407 245 (1985).

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MODULATION OF ELASTIN PRODUCTION

241

Effects of Extracellular Matrices on Elastin Production and Accumulation The progressive "dedifferentiation" of elastin-producing cells in culture suggests a phenotypic instability resulting from removal of cells from their in vivo environment. In terms of elastin biosynthesis, there are three clear differences between ligament cells grown on tissue culture plastic and cells in viable ligament tissue (i.e., cells surrounded by extracellular matrix). (I) Cells in ligament minces rapidly incorporate soluble elastin into an insoluble polymer, whereas elastin precursors synthesized by cultured cells remain soluble. (2) Cells in tissue minces synthesize more elastin per cell than do ligament cells in culture. (3) When tissue minces are incubated with flAPN to inhibit cross-linking, soluble elastin is not released into the culture medium but remains associated with the matrix. In contrast, >90% of the soluble elastin synthesized by ligament cells in culture is released into the medium. The microenvironment of cells growing on tissue culture plastic is far different from the intraceUular matrix of the intact tissue and it has been suggested by many studies that culturing cells on biological matrices is more conducive to proper phenotypic expression. When fully differentiated ligament cells are grown on slices of intact ligament, two changes are observed: (1) total elastin production increases 2- to 3-fold and approximates elastin synthesis by cells in intact tissue, and (2) a large percentage of newly synthesized elastin remains associated with the cell layer and becomes cross-linked. Addition of/3APN prevents desmosine accumulation but alters only slightly the distribution of elastin between the medium and cell layer (Fig. 2). These results show that ECM contributes to the specific differentiated properties of elastin-synthesizing cells and promotes proper fiber organization and cross-linking. Similar results are obtained using ECM from cultured cells but the amount of tropoelastin that remains associated with the cell layer is variable and cross-linking of tropoelastin chains is less efficient.

Preparation of Acellular ECM from Ligament Tissue Fetal bovine ligament tissue is repeatedly freeze-thawed to kill ligament fibroblasts. Loss of tissue viability is confirmed by incubating a section of tissue for 6 hr with [3H]leucine and demonstrating no trichloroacetic acid-precipitable radioactivity in the incubation medium. Nonviable tissue is then serially sectioned tangential to the long axis of the ligament on a freezing microtome to produce 80- to 120-tzm-thick slices.

242

MAJOR C O M P O N E N T S OF T H E E X T R A C E L L U L A R M A T R I X

I0 Z _(2 i-

8

ca Oca

6

[] Cell Extract [] Medium

[12]

T

~ Z ~4 <

..A Is/

2 Plastic ECM t__ -~'APN

Plastic J

~--

ECM

+/~APN---J

FIG. 2. Distribution of tropoelastin between medium and cell layer in first-passage ligament cells grown on tissue culture plastic or on ligament ECM. Cells were grown for 4 days in the presence or absence of/3APN. Elastin levels in cell culture medium and in an acetic acid extract of the matrix cell layer were determined by radioimmunoassay. Values are the mean plus standard deviation of triplicate values of duplicate plates. Reproduced from The Journal of Cell Biology (1984) 98, 1804-1812 by copyright permission of The Rockefeller University Press.

Tissue slices are spread o v e r the bottom of tissue culture dishes and sterilized by gas sterilization or by rinsing with 70% alcohol and drying overnight under a germicidal lamp. Freshly trypsinized cells can then be plated onto the ligament slices and cultured as described above. Cells can be r e m o v e d from the ligament matrix by trypsinization.

Preparation o f E C M from Cultured Cells One o f the gentlest methods for isolating ECM produced by cultured cells is hypotonic shock. The advantage o f this approach is that the buffers used are mostly nonextractive so that ECM ultrastructure remains relatively well intact. The disadvantage is that the cellular material (membranes, cytoskeletal components, etc.) remains associated with the cell layer. In addition, cells that elaborate an extensive ECM (such as chondrocytes and rat aortic smooth muscle cells) are difficult to disrupt using this technique. E C M p r o d u c e d by confluent cells is denuded by washing the cell layer with sterile 50 m M H E P E S , p H 7.4, supplemented with protease inhibitars followed by incubating the cell layer at 37 ° for 10-20 min in the same buffer. The lysis buffer is removed by aspiration after gentle swirling and is replaced with growth medium. Trypsinized cells are then added to the dish and cultured as described above. Alternatively, ECM can be isolated

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MODULATION OF ELASTIN PRODUCTION

243

by washing the cell layer with 0.02 M NH4OH in distilled water. 2° When base treatment is used, confluent cultures are first washed with distilled water and then exposed to NHaOH for 5 min, followed by washing with PBS or culture medium. Fewer nuclei or cytoskeletal elements remain after base treatment. Extracellular matrices can also be denuded by treatments with chaotropic agents or detergents. While these reagents are effective at removing cellular material, they also extract, to varying degrees, macromolecules from the extracellular matrix. One of the least extractive methods is treatment with urea. 21 Confluent cultures are washed twice with DMEM and then exposed for 10-20 min at 37° to DMEM supplemented with 2 M urea and 0.5% fetal calf serum. Medium containing the detached cells is aspirated (at no time are cells pipetted up and down). The gentle washing process is repeated four times. Once the matrix is free of cells, plates are rinsed three times with PBS. Treatment of cell cultures with detergents is most disruptive, but even between the detergents there are gradients of extraction. It has been our experience with fibroblasts that octyl-fl-glucoside is the least extractive detergent followed by Triton X-100 and deoxycholate. Soluble Modulators of Elastin Synthesis Steroid H o r m o n e s

Several studies have suggested that glucocorticoids stimulate elastin production both in vitro and in vivo 4,2z and that steroids regulate the onset of elastogenic differentiation during development 23 or modulate the expression of different elastin gene products, z4 Using cultured ligament cells, we have shown that glucocorticoids stimulate elastin synthesis in elastin-producing cells but do not induce elastin production in undifferentiated cells. 1~ Stimulation of elastin production in differentiated ligament cells is reversible and dose dependent, with maximal stimulation occurring at physiological concentrations of the natural bovine glucocorticoid, cortisol, as well as the synthetic glucocorticoids dexamethasone and methyl2o D. Gospodarowicz, K. Hirabayashi, L. Giguere, and J. P. Tauber, J. Cell Biol. 106, 568

(1981). 21 D. Gospodarowicz, R. Gonzales, and D. K. Fujii, J. Cell. Physiol. 114, 191 (1983). z2 W. Burnett, R. Eichner, and J. Rosenbloom, Biochemistry 199 1106 (1980). 23 R. Eichner and J. Rosenbloom, Arch. Biochem. Biophys. 198, 414 (1979). 24 L. L. Barrinean, C. B. Rich, A. Przybyla, and J. A. Foster, Dev. Biol. 87, 46 (1981).

244

MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

300

[12]

• o.o~°,~°,oo~

_--

o

~/J Hydrocortisone 1"7 Methylprednisolone

~.~ ~0 200

0

Io3

a65

16r

169

16'

16~

MOLAR CONCENTRATIONADDED FIG. 3. Late log phase FCL-270 cells (second passage) were incubated with dexamethasone, hydrocortisone, or methylprednisolone for 24 hr. Elastin levels in the medium and cell layer (combined) were determined by radioimmunoassay. Results, expressed as percentage change from control (untreated cells), represent the mean and standard deviation of duplicate determinations from triplicate plates at each hormone concentration. Reproduced from The Journal of Biological Chemistry (1984) 259, 12414-12418 by copyright permission of the American Society of Biological Chemists, Inc.

prednisolone. The response to dexamethasone, however, is generally 2to 4-fold greater than to the other two hormones (Fig. 3). Glucocorticoid stimulation is time dependent with a lag period of 8-12 hr before increased tropoelastin synthesis is observed. Ligament cells grown in the

tcGMP

/ _ C a 2+

\ + C a 2+

I

l No Response

Elevated Elastin Production

tcAMP

Terminates cGMP Stimulation

FIG. 4. Schematic illustration of the interrelationships between cGMP and cAMP regulation of elastin production. Agents that elevate cGMP levels in intact cell systems stimulate elastin synthesis in the presence of extracellular calcium. Elastin synthesis is unaffected, however, by changes in cAMP. When cAMP and cGMP levels are raised together, cGMP stimulation of elastin production is blocked by cAMP in a dose-dependent fashion, suggesting that the degree of cGMP-dependent stimulation of elastin synthesis is inversely related to the cAMP concentration.

[12]

MODULATION OF ELASTIN PRODUCTION

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presence of 10 nM dexamethasone have a slower population doubling time than cells grown without hormone. The responsiveness of ligament cells to glucocorticoid stimulation is highly dependent on a number of cell culture conditions. Maximal stimulation is obtained with early-passage cells, at the late log phase of growth, when exposed to dexamethasone in the presence of greater than 3% (v/v) fetal bovine serum. While fetal bovine serum seems to be required to optimize hormone stimulation, there can be a variable response associated with different serum batches. The properties of serum responsible for this variability are unknown, although endogenous levels of steroid hormones in serum are important considerations in this regard (see Table I). In 71 lots of fetal calf serum produced and assayed by Hyclone Laboratories, Inc., cortisol values varied 30-fold between 0.1 and 3.0/xg/dl, with a mean value of 0.7 p,g/dl. Cortisol levels in calf serum were substantially higher, averaging 4.9 /~g/di (values provided by Hyclone Laboratories, Inc., Logan, Utah).

Cyclic Nucleotides Numerous extracellular signals produce physiological stimuli in target cells by increasing intracellular levels of the prominent second messengers, cAMP, cGMP, and calcium. Evidence that connective tissue production is under the regulatory influence of cyclic nucleotides is supported by the finding that, in some cell types, increases in intraceUular levels of cAMP result in decreased collagen production and enhanced proteoglycan synthesis. Agents that promote the effects of cGMP in intact cell systems stimulate elastin synthesis in ligament cells in vitro. These changes, however, are dependent on the continued presence of intracellular calcium, since ligament cells incubated in calcium-free medium or with the calcium ionophore A23187 are no longer responsive to stimulation by cGMP derivatives and produce substantially less elastin than control ceils. 25 In contrast to the specific stimulatory effect on elastin production by cGMP, elastin synthesis by ligament cells is unaltered by changes in intracellular cAMP levels. When cGMP and cAMP derivatives are administered together, however, cGMP stimulation of elastin production is blocked by cAMP (Fig. 4). Thus, an imbalance in the concentration of the cyclic nucleotides in favor ofcGMP leads to a positive, stimulatory signal that is reversed by cAMP. When both cyclic nucleotides change in the 25 R. P. Mecham, B. D. Levy, S. L. Morris, J. G. Madaras, and D. S. Wrenn, J. Biol. Chem. 260, 3255 (1985).

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[13]

same direction, their effects are likely to be antagonistic with no net change in elastin production. In this way, cAMP could regulate the responsiveness of the cell to extracellular signals by adjustments in sensitivity to cGMP stimulation; high intracellular concentrations of cAMP could "desensitize" or render the cell unresponsive to extracellular signals that require cGMP mediation. Acknowledgments This work was supported by National Institutes of Health Grants HL-26499, HL-29594, and Training Grant HL-07317.

[13] I m m u n o l o g y o f E l a s t i n

By DAVID S. WRENN and ROBERT P. MECHAM Antisera to soluble and insoluble elastin have been used to estimate the amount of elastin in tissue samples, serum, and other bodily fluids, to quantify and immunoprecipitate elastin synthesized by cell and organ cultures, as well as to localize elastin in intact tissues and cells. This chapter will describe methods for the preparation of polyclonal and monoclonal antibodies to insoluble or solubilized elastin and will detail their use in a variety of immunological procedures. Purified, mature elastin is a weak antigen and antisera of relatively low titer are obtained when the insoluble protein, elastin-derived peptides prepared by digestion of insoluble elastin with alcoholic potassium hydroxide (x-elastin), or oxalic acid-solubilized elastin (a-elastin) are used as antigens. This problem is compounded by the fact that antibodies to these antigens are generally nonprecipitating, presenting difficulty in determining precise antibody titers. In the past, failure to detect significant antielastin antibodies in immune sera was attributable in part to the relatively insensitive methods used to test the antisera. With the advent of improved immunodetection techniques, however, useful polyclonal and monoclonal antielastin antibodies have been generated in a variety of animal species using fine suspensions of insoluble elastin, a-elastin, r-elastin, and pancreatic or neutrophil elastase digests of insoluble elastin as antigens. 1 1R. P. Mecham and G. Lange, this series, Vol. 82, p. 744.

METHODS IN ENZYMOLOGY, VOL. 144

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved,