REGULATION OF EXTRACELLULAR MATRIX PROTEINS BY TRANSFORMING GROWTH FACTOR β1IN CULTURED PULMONARY ENDOTHELIAL CELLS

Cell Biology International 1999, Vol. 23, No. 1, 61–72 Article No. cb980325, available online at http://www.idealibrary.com on

REGULATION OF EXTRACELLULAR MATRIX PROTEINS BY TRANSFORMING GROWTH FACTOR â1 IN CULTURED PULMONARY ENDOTHELIAL CELLS GOURI SHANKER, DAN OLSON, ROGER BONE† and RAJINDER SAWHNEY* Department of Surgery, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284, U.S.A. Received 8 April 1998; accepted 12 October 1998

Transforming growth factor beta-1 (TGF-â1), which is present in lung tissue, has been suggested to play a role in modulating vascular cell function in vivo. The action of TGF-â1 in vivo, especially at the local site of application to connective tissue, is anabolic and leads to pulmonary fibrosis and angiogenesis, strongly indicating that TGF-â may have practical applications in repair of tissue injury caused by burns, trauma, or surgery. In the present study, we have used cultured bovine pulmonary artery endothelial (BPAE) cells as a model system. Expression of various proteins, including SPARC (secreted protein acidic and rich in cysteines), type IV procollagen and fibronectin (FN) was examined by radiolabeling the cells with [3H]proline, immunoprecipitation with specific antibodies, and Northern blot analyses by using specific cDNA probes. Cultured cells were labeled with [3H]proline for 24 h in either the absence or in the presence of TGF-â1 (0–20 ng/ml). Incorporation of radioactivity was observed in a concentration-dependent manner, maximal at 5 ng/ml. Northern blot hybridization demonstrated that TGF-â1 (5 ng/ml) treatment of BPAE cells caused an increase in steady-state levels of mRNA for SPARC (75%) and FN, thus, indicating a positive modulation. To determine whether TGF-â1 has an effect on cell proliferation in the cultures, the incorporation of [3H]thymidine into cellular nuclei was used to measure DNA replication; 5 ng/ml had an inhibitory effect (42%) on cell proliferation. Protein production by TGF-â1, surprisingly, showed decrease in SPARC levels (42%) and in contrast increased levels of FN (86%) and type IV procollagen (334%). Our results indicate that on exposure to TGF-â1, cultured BPAE cells differentially change expression of extracellular matrix proteins which may be important in  1999 Academic Press remodeling of tissue and healing at sites of injury. K: TGF-â1; gene regulation; bovine pulmonary endothelial cells; SPARC; collagen; fibronectin.

INTRODUCTION Despite various reports on the functional aspects of TGF-â1, very little is known about its regulation of the extracellular matrix (ECM) genes in arterial endothelium. Endothelial cells, one of the crucially important components of the vascular system, are known to play an important role in many vital and biologically prominent processes such as tissue growth, development and repair. One of the important *To whom correspondence should be addressed. †Deceased. This paper is dedicated in honor and memory of Dr Roger C. Bone. 1065–6995/99/010061+12 $30.00/0

functions of the endothelial cells is to synthesize and secrete a variety of ECM proteins which help to make up the extracellular environment. Various studies have demonstrated that cellular functions and responses can be affected by the surrounding ECM, and it is also well established that ECM plays a crucial role in maintaining the structural as well as functional integrity of cells (Sage and Bornstein, 1991; Sawhney et al., 1997; Yao and Eghbali, 1992). Keeping all these facts in mind, it becomes clear that any alterations in the balanced production and breakdown of ECM components could result in a pathologic condition (Sporn et al., 1986).  1999 Academic Press

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Therefore, a systematic elucidation of the mechanisms by which the ECM may be altered would be of importance as well as of interest in both health and disease situations. Two types of transforming growth factors (TGF-á and TGF-â) have been purified and characterized. TGF-â is a family of five structurally related molecules (TGF-â1–â5). This family of hormone-like polypeptides participates in the control of growth differentiation and morphogenesis in cultured cells and organisms from insects to mammals (Goustin et al., 1986; Massague, 1987; Sporn et al., 1987). TGF-â1 is secreted by many non-transformed and transformed cells in a biologically inactive form (Lawrence et al., 1984a,b; KryceveMartinerie et al., 1985) that is apparently unable to bind to the TGF-â receptor (Wakefield et al., 1987). Activation of this circulated latent form by exogenous proteases or by the acidic microenvironment in healing wounds may be an important regulatory mechanism of TGF-â action. Although many cells, including endothelial cells (Miyazono et al., 1994), can synthesize TGF-â1, a major source is the alpha granule of platelets (Massague, 1987; Sporn et al., 1987), which is consistent with its implicated role in wound healing (KryceveMartineri et al., 1985). TGF-â is known to act by both autocrine and paracrine mechanisms (Brattain et al., 1993) as a multifunctional regulator of cellular activity, it inhibits the proliferation of many epithelial and tumor cells (Ranchalis et al., 1987), smooth muscle cells (Asoian and Sporn, 1986; Owens et al., 1988; Lawrence et al., 1994), human umbilical vein rat heart and bovine aortic and capillary endothelial cells (Baird and Durkin, 1986; Frater-Schroder et al., 1986; Heimark et al., 1986; Takehara et al., 1987; Nugent and Newmann, 1989) and stimulates the growth of a variety of fibroblastic cell type (Sporn et al., 1987) and osteoblasts (Centrella et al., 1987; Robey et al., 1987). Many mesenchymal and epithelial cells respond to TGF-â1 with elevated expression of fibronectin, various types of collagen and other cell-adhesion molecules (Ignotz and Massague, 1986, 1987; Sporn et al., 1987; Yoshida et al., 1992). A recent study from our laboratory has shown that TNF-á downregulates the expression of fibronectin and upregulates collagen IV gene in cultured bovine pulmonary artery endothelial (BPAE) cells (Yao et al., 1995). A study using 3T3 adipocytes, showed that the TNF-á effect may be mediated by TGF-â, as the levels of the latter are reported to show increase in response to TGF-á (Weiner et al., 1989). To test the hypothesis that the

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mechanism of regulation of extracellular matrix proteins in endothelial cells may be similar, we carried out the present study in which we examined the effect of TGF-â1 in the synthesis of total extracellular (and intracellular) proteins and specifically, on the regulation of SPARC, FN and collagen in cultured BPAE cells. The results showed that the TGF-â1 caused an increase in steady-state levels of mRNA for SPARC and FN, thus indicating a positive modulation. However, protein production by TGF-â1 showed decrease in SPARC levels and in contrast, increased levels of FN and type IV procollagen. Thus, our studies indicate that TGF-â1 differentially regulates the expression of ECM proteins in BPAE cells and the mechanism for regulation of FN may be different from that of TNF-á.

MATERIALS AND METHODS Cell culture and treatment The BPAE cells used in these studies were isolated by enzymatic digestion from fresh bovine heart– lung preparations, as described earlier (Sawhney et al., 1992; Yao et al., 1995). The cells were cultured in medium RPMI 1640 supplemented with 10% fetal bovine serum at 37C and under 5% CO2/95% humidified air. Confluent cells from passages 6 or 7 were used for all studies. Preparatory to experimental treatments, BPAE cells were plated at a density of 4–5105 per 100 mm culture dish or 2–3105 per 60-mm dish and grown until confluent in the serum medium. Cells were then washed once with serum-free culture medium and incubated in serum-free medium for up to 24 h. The serum-free medium was replaced before experimental treatments. Various concentrations of recombinant human TGF-â1 (GIBCO-BRL, Gaithersburg, MD, U.S.A.) or equal volumes of vehicle (0.1% bovine serum albumin in PBS) were added to the serum-free culture medium for different periods of time to form the treatment and control conditions. RNA extraction Total RNA was extracted from confluent cell layers using a modification of Chomczynski and Sacchi’s method (Chomczynski and Sacchi, 1987) using TRI reagent (MRC Inc., Cincinnati, OH, U.S.A.). Total RNA was quantified by absorbance at 260 nm.

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Northern blot analysis Steady-state mRNA levels were determined by Northern hybridization analysis (Yao et al., 1995). In general 10–15 ìg of total RNA were heatdenatured in a mixture of 50% formamide, 2.2  formaldehyde, 0.53-[N-morpholino] propanesulfonic acid (MOPS) buffer (pH 7.0). The RNA was then electrophoresed in a 1% agarose gel, transferred to a Gene-Screen Plus membrane (New England Nuclear, Boston, MA, U.S.A.), in a 10SSC buffer and baked for 2 h at 80C in a vacuum oven. The blots were prehybridized for 6–8 h in a mixture of 5sodium chloride/sodium citrate (SSC), 0.1% SDS, 1Denhardt’s solution, 50% deionized formamide, 0.05  phosphate buffer (pH 7.0), and 100 ìg/ml salmon testes DNA and then hybridized with labeled probe for 36–40 h at 40C. Hybridization was performed in a buffer solution containing enough radioactive probe to give the hybridization medium a scintillation count of 3–5106/min/ml. The cDNAs were radioactively labeled by the random primer DNA labeling system according to the manufacturer’s instructions (GIBCO-BRL). Complementary DNAs were labeled to a specific activity of 1109 counts/ min/ìg with a [32P]dCTP (specific activity of 3000 Ci/mmol; Amersham). The labeled probes were heated to 95C for 5 min and immediately cooled to 4C before adding to fresh prehybridization solution. The recombinant plasmid cDNA used as probes were as follows: pG43 (mouse SPARC cDNA, a gift of Dr B. Hogan); pFH154 (human FN cDNA, American Type Culture Collection; and HHCSA 65 (18S rRNA, American Type Culture Collection). After hybridization, the membranes were washed using procedures that have previously been described (Yao et al., 1995) and then exposed to Kodak X-ray film at
70C. Incorporation of [3H]thymidine Cultures of BPAE cells were grown until confluent in medium supplemented with 10% FBS on 60-mm culture dishes. After making cells serum-free for up to 24 h, TGF-â1 (5 ng/ml) was added to the treatment dishes containing fresh serum-free culture medium. This treatment lasted for 20 h and was followed by the addition of [3H]thymidine (specific activity of 87 Ci/mmol; Amersham) to give the medium a specific activity of 5 ìCi/ml. After 4 h, the cells were processed as described earlier and the radioactivity incorporated in DNA was estimated by 10% TCA precipitation as described by Nugent and Newmann (1989). DNA was quantitated by a

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previously described colorimetric method (Giles and Myers, 1965). Phase contrast microscopy To examine the morphological changes occurring in BPAE cells after 24 h exposure to TGF-â1, phase contrast micrographs were taken in both treated and non-treated dishes using Olympus phase contrast microscope. Metabolic labeling Cultured BPAE cells were grown until confluent in medium containing 10% FBS in 60 mm dishes. Cells were placed in serum-free media supplemented with 50 ìg/ml ascorbic acid and 50 ìg/ml â-aminopropionitrile. Control and treatment (various concentrations of TGF-â1 in the range of 0.1–20 ng/ml) cells were then labeled with 50 ìCi/ml of -[2,3,4,5-3H]proline (81 Ci/mmol; Amersham) for 24 h. The radiolabeled medium of the culture was harvested into a cocktail of protease inhibitors (final concentration: 10 m N-ethyl maleimide (MalNEt), 5 m EDTA, and 2 m PMSF). The medium was centrifuged at 400g for 15 min at 4C. The supernatant was immediately placed in
80C storage until use. The cell layer, after two washings with cold PBS, was lysed in buffer (50  Tris (pH 8.0), 1% NP-40, 150 m sodium chloride, 5 m MalNEt, 5 m EDTA, and 2 m PMSF) and centrifuged at 12,000g for 10 min at 4C. The supernatant was mixed with an equal volume of glycerol and stored at
20C. To measure the newly synthesized total protein released into the culture medium, aliquots containing appropriate quantities of radiolabeled protein were precipitated with 20% cold TCA and filtered. After washing with 10% TCA, the radioactive protein was quantitated by liquid scintillation counting. The protein content, in appropriate aliquots of media and cell lysate, was estimated by using Bio-Rad’s DC protein assay kit II, which is based on a modification of Lowry protein assay. The specific incorporation of [3H]proline in the total proteins was calculated on a per-microgram basis of protein. Immunoprecipitation and SDS-PAGE Appropriate aliquots (containing equal quantities of TCA-precipitable radioactivity from treated and control cultures) of radiolabeled medium or cell lysate were mixed with equal volumes of buffer 1 (50 m Tris/Cl (pH 8.0), 1% NP-40, 400 m

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sodium chloride, 5 m EDTA, and 1 m PMSF) containing the specific antiserum. The specific antibodies used were as follows: rabbit anti-bovine

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collagen type IV antibody (Sawhney et al., 1997); rabbit anti-bovine antibody LF-Bon I (a gift from Dr Larry Fisher, National Institutes of Health) and rabbit anti-bovine FN antibody (Calbiochem, La Jolla, CA, U.S.A.). The mixture was incubated at 4C for 6 h and subsequently agitated overnight with 60 ìl (6 mg) of protein A-Sepharose (Sigma) suspension at 4C (Sawhney et al., 1991). The sepharose beads were collected by centrifugation at 850g for 10 min at 4C. They were washed five times with buffer 1 and once with 10 m Tris/ EDTA, pH 6.8. The antigens were eluted by boiling for 5 min in 60 ìl of 2Laemmli sample buffer and then isolated by centrifugation at 1500g for 10 min through Bio-Rad polypropylene columns (Sigma). The radioimmunoprecipitated samples were denatured by heat and electrophoresed on a polyacrylamide gel (4% for stacking gel and 6.5% for main gel). Following the processes of fluorography (Bonner and Laskey, 1974) and drying, Kodak X-ray films were exposed to the gel at
70C using intensifying screens. Quantification and data analysis The mRNA signals on the Northern autoradiograms and protein signals from the radioimmunoprecipitated polypeptides were quantitatively analyzed by laser densitometry (de Leeuw et al., 1989), as described previously (Yao and Eghbali, 1992). To determine the abundance of the various mRNAs, ribosomal RNA bands transferred to the Gene-Screen Plus membrane were made visible by ethidium bromide staining and recorded with photography. We have used the 18S band for normalizing other RNA bands; this form of internal control is an accepted method for determining the relative quantity of specific RNAs (de Leeuw et al., 1989; Yao and Eghbali, 1992). The density of the 18S ribosomal RNA band was measured by densitometric scanning of the photographic negatives. In order to obtain a precise measure of mRNA per unit of total RNA (i.e. to normalize the mRNA reading), the density of individual mRNA bands was divided by the density of the corresponding 18S ribosomal RNA band. Samples from treated and corresponding control groups were always run Fig. 1. Appearance of BPAE cells that were grown in the absence or presence of TGF-â1 by phase contrast microscopy. Cells were grown in the medium containing 10% fetal bovine serum until confluency. After switching to serum-free medium, the cells were incubated in the absence (a) or presence (b) of TGF-â1 (5 ng/ml) for 24 h before taking the photographs (100 magnification).

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Fig. 2. A dose-response study on the effect of TGF-â1 on total protein synthesis in medium of cultured BPAE cells. Confluent, quiescent cells were labeled with [3H]proline in either the absence or presence of various concentrations of TGF-â1 (0–20 ng/ml) for 24 h. The radioactivity of TCA-precipitated aliquots of culture medium from control and treated cells was represented as percentage change in [3H]proline incorporation, as described in the Methods. Data are expressed as meanSE (N=3). The specific incorporation of [3H]proline in the total proteins was calculated on a per-microgram basis of proteins. *P<0.01, **P<0.001, ***P<0.05.

on the same gel. All quantitative data presented represent the results of at least two individual experiments and are displayed as meanstandard error (SE). The Student’s t-test was used in the statistical analysis of the data and a significant result was recorded when the P value was less than 0.05.

RESULTS Effect of TGF-â1 treatment on the morphology of bovine pulmonary artery endothelial cells We examined the effect of TGF-â1 on the possible changes in morphology of BPAE cells. The cells were grown as described in detail in Methods until confluent. Subsequently, cells were switched to serum-free media for 24 h after which some of the dishes were treated with TGF-â1 (5 ng/ml) for 24 h before taking the photographs in a phase contrast microscope of both treated and untreated (control) cells. As shown in Figure 1, there were significant changes in the morphology of the cells as the treatment of BPAE cells with TGF-â1 caused a

5

15 10 TGF-β1 (ng)

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Fig. 3. A dose-response study on the effect of TGF-â1 on total protein synthesis in cell lysates by cultured BPAE cells. Confluent, quiescent cells were labeled with [3H]proline in either the absence or presence of various concentrations of TGF-â1 (0–20 ng/ml) for 24 h. The radioactivity of TCA-precipitated aliquots of cell lysates from control and treated cells is represented as percentage change in [3H]proline incorporation, as described in the Methods. Data are expressed as meanSE (N=3). The specific incorporation of [3H]proline in the total proteins was calculated on a per-microgram basis of proteins. *P<0.02, **P<0.001.

change from a typical ‘cobblestone’ morphology (Fig. 1a) to a large and flattened type of cell (Fig. 1b). Effect of TGF-â1 on total protein changes as well as specific protein changes in media and cell lysates In order to evaluate whether the nascently synthesized as well as media-released protein production is altered by TGF-â1, BPAE cells were metabolically labeled with [3H]proline under appropriate experimental conditions (as described in detail in Methods) in the presence of different concentrations (0–20 ng/ml) of TGF-â1 for 24 h. The total protein production was estimated by measuring the radioactivity of TCA-precipitated aliquots of culture media as well as cell lysates and representing it as cpm/ìg protein. For both media (Fig. 2) as well as cell lysate (Fig. 3), there was a TGF-â1 dose-dependent increase in incorporation with a peak at approximately 5–10 ng/ml after which it declined. To further analyze changes in the levels of specific proteins like FN, type IV procollagen and SPARC, these proteins were quantitatively immunoprecipitated from both culture media and

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Fig. 4. SDS-PAGE analysis of immunoprecipitated fibronectin from culture medium. Confluent, quiescent BPAE cells were labeled for 24 h with [3H]proline in the presence of TGF-â1 (5 ng/ml) or vehicle. Fibronectin was immunoprecipitated from aliquots of culture medium containing equal amounts of TCA-precipitable radioactivity. Lane 1 shows 14C-labeled protein molecular weight standards. Lane 2 represents control cells and lane 3 shows results from TGF-â1-treated cells. The left panel is representative fluorogram of three individual experiments; right panel presents the results of densitometry scanning of the fluorogram. Data are expressed as meanSE of three individual experiments.

cell lysates (of control and TGF-â1 treated cells) with corresponding specific antisera. The precipitates were quantitatively analyzed by densitometric scanning of SDS/PAGE fluorograms. The results showed that there was an increase in FN production and secretion in culture media (74%, Fig. 4) and cell lysate (12%, data not shown) in TGF-â1-treated cells compared with

untreated control cells. Furthermore, type IV procollagen production and secretion into the culture medium and in cell lysate was also increased 312% and 22%, respectively, by TGF-â1 (Fig. 5). However, SPARC production and secretion into media, in contrast to FN and type IV procollagen, was found to be decreased by 42% in TGF-â1 treated cells (Fig. 6).

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Fig. 5. SDS-PAGE analysis of immunoprecipitated type IV procollagen. Confluent, quiescent BPAE cells were labeled for 24 h with [3H]proline in the presence of TGF-â1 (5 ng/ml) or vehicle. Type IV procollagen was immunoprecipitated from aliquots of culture medium or cell lysate containing equal amounts of TCA-precipitable radioactivity. Lane 1 shows results from control cells (medium). Lane 2 represents results from TGF-â1-treated cells (medium) and lane 3 shows results from control cells (cell lysate) and lane 4 shows results from treated cells (cell lysate). Lane 5 shows 14C-labeled type I collagen molecular weight standard.

Effect of TGF-â1 on steady-state levels of mRNA in BPAE cells Steady-state levels of SPARC and FN mRNA were determined by blotting the total RNA from BPAE cells treated with TGF-â1 or control into genescreen plus membranes and then hybridizing with specific cDNA probes for SPARC, FN and 18S rRNA. Northern hybridization analysis indicated that TGF-â1 treatment of BPAE cells caused an increase in SPARC mRNA levels as well as FN

mRNA levels (Fig. 7). A dose–response study of the effect of TGF-â1 on the induction of SPARC mRNA was carried out by treating BPAE cells with different concentrations of TGF-â1 and then running Northern blot on isolated RNAs. The results (Fig. 8) obtained through densitometric scanning of autoradiograms showed a dose-dependent increase in SPARC mRNA levels with a peak seemingly at around 10 ng/ml TGF-â1 concentration (Fig. 8; upper panel). We also quantified the mRNA for SPARC in the cells after 24 h treatment

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(a) TGF-β1 (ng/ml) 0 Lane 1

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18S rRNA

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Fig. 7. Northern blot hybridization analysis of total RNA from cultured BPAE cells. Total RNA (10 ìg) from confluent, quiescent cells was incubated with vehicle (control) or various concentrations of TGF-â1 (5, 10 and 20 ng/ml) for 24 h and subjected to size fractionation by electrophoresis on 1% agarose gel. The methods used in RNA transfer and blot hybridization are described in the Methods. Lanes of autoradiograms containing mRNAs hybridized with specific cDNA probes are shown. (a) SPARC mRNA and 18S rRNA, lane 1 (control), lane 2 (TGF-â1; 5 ng/ml); lane 3 (10 ng/ml), and lane 4 (20 ng/ml). (b) Levels of fibronectin mRNA (control; lane 1) or 10 ng/ml TGF-â1 (lane 2).

Fig. 6. SDS-PAGE analysis of immunoprecipitated SPARC from culture medium. Confluent, quiescent BPAE cells were labeled for 24 h with [3H]proline in the presence of TGF-â1 (5 ng/ml) or vehicle (control). SPARC was immunoprecipitated from aliquots of culture medium containing equal amounts of TCA-precipitable radioactivity. Lane 1 shows 14 C-labeled protein molecular weight standards. Lane 2 represents results from control cells and lane 3 shows results from TGF-â1-treated cells. The analyses were performed by SDSPAGE fluorography as described in Materials and Methods.

with 5 ng/ml of TGF-â1. The abundance of mRNA was found increased by 75% (Fig. 8; lower panel). Effect of TGF-â1 on cell proliferation in BPAE cells In order to determine the effect of TGF-â1 on cell proliferation, we analyzed the incorporation of

[3H]thymidine into cellular nuclei as a measure of DNA replication. The results showed (Fig. 9) that when confluent, quiescent cells were treated with TGF-â1 for 24 h, there was an inhibition (42%) of endothelial cell proliferation.

DISCUSSION TGF-â1 is known to be a major modulator of expression of connective tissue genes (Massague, 1990). This fact has been supported from the studies that TGF-â1 causes fibrosis at the site of injection (Roberts et al., 1986) and also from various other studies where the expression of many extracellular matrix constituents was found to be elevated at both protein and mRNA levels when treated with TGF-â1 (Ignotz and Massague, 1986; Fine and Goldstein, 1987; Varga et al., 1987; Ogata et al., 1997). A critical role for TGF-â in

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2100 [ H]Thymidine incorporation (cpm/µg DNA)

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Fig. 9. [3H]Thymidine incorporation into the cell nuclei of control and TGF-â1-treated BPAE cells. Confluent quiescent cells were treated with TGF-â1 (5 ng/ml) for 24 h. The amount of methyl [3H]thymidine incorporated per microgram of DNA in the cell lysates was determined as described in Materials and Methods. Data are represented as meanSE (N=9 in each group). *P<0·001.

TGF-β1 (ng/ml)

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Fig. 8. Densitometric evaluation of Northern blot hybridization results for SPARC mRNA. The autoradiogram obtained in Figure 7 was densitometrically scanned to quantitate SPARC mRNA bands and the corresponding 18S rRNA as described in Materials and Methods. Quantification of mRNA for SPARC (and the effect of various concentrations of TGF-â1 on its abundance) in BPAE cells is shown (a) whereas (b) shows quantitative SPARC mRNA levels when cells were treated with 5 ng/ml of TGF-â1. Data are presented as meanSD of two individual experiments (Control 0.50.16; treatment 0.880.34; P<0.3).

pulmonary inflammation was demonstrated in a model of bleomycin-induced lung injury (Khalil et al., 1989). TGF-â1 is multifunctional, since it can either stimulate or inhibit proliferation, and can either stimulate or inhibit other essential processes in cell function.

Interestingly, TNF-á cytokine’s response was studied by Weiner et al. (1989) using 3T3-L1 adipocytes where they showed that it increases TGF-â mRNA content in these cells. Sato and Rifkin (1989) from their studies have suggested that TGF-â may mimic some of the actions of TNF-á. Therefore, on the basis of above observations and also based on the premise that some of the actions of TNF-á may be mediated by TGF-â, we have carried out the present studies to examine the TGF-â1 regulated expression of certain distinct extracellular matrix components in BPAE cells. The results from our studies demonstrate an increase in the production of newly synthesized total proteins in response to TGF-â1 in a dosedependent manner, in both culture media as well as cell lysates. On examining the effect of TGF-â1 on cell proliferation, it was found to inhibit cell growth. Other investigators have also noticed inhibition in proliferation of endothelial cells to a varying degree under somewhat different conditions, in response to TGF-â (Heimark et al., 1986; Baird and Durkin, 1986; Frater-Schroder et al., 1986). Among the extracellular matrix proteins examined, levels of fibronectin and procollagen á1 (IV) were found to be increased in response to TGF-â1. These data are consistent with the concept that TGF-â is an important mediator of tissue repair, while in contrast, the level of protein SPARC (Sawhney, 1995) was found to be

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decreased. It is likely that in the present study concomitant inhibition of SPARC and stimulation of FN and procollagen á1 (IV) proteins, have a functional role in matrix remodeling. It is tempting to speculate that TGF-â is affecting the cell membrane of endothelial cell as a result of which most of the FN is secreted out. In case of type IV collagen, it is likely that TGF-â is increasing the synthesis of the enzyme proline hydroxylase, which enhances hydroxylation and makes type IV collagen more secretory as shown in periacinar fibroblastoid cells (Kato et al., 1996). On examining the mRNA levels for FN and SPARC, both were found to be increased in BPAE cells when challenged with TGF-â1. In case of SPARC, regulation by TGF-â may be via a post-transcriptional mechanism involving stability, processing of nascent SPARC transcripts or transport of mRNA into the cytoplasm (Turnbull and Lightowlers, 1998). On the basis of Northern blot analysis, there is no evidence that TGF-â regulates alternative splicing or polyadenylation of SPARC mRNA. There is another mechanism by which accumulation of SPARC mRNA could occur: TGF-â1 may render the RNA non-functional while still allowing it to efficiently hybridize with the cDNA probe. The most often cited mechanisms by which protein production is controlled involve alterations in transcription (Shanker and Sawhney, 1996), translation and post-translational modifications (Sawhney and Bone, 1993). Our results are in contrast with earlier report that expression of SPARC was not altered by TGF-â in osteoblastic bone cells (Wrana et al., 1991). Ignotz et al. (1987) have reported that, in NRK rat fibroblasts, the TGF-â mediated increase in FN and collagen mRNA was observed at the level of transcription. The phenomenon of differential regulation of ECM protein gene expression in BPAE cells, in response to TNF-á was also observed in our recently published studies (Yao et al., 1995). TNF-á downregulated expression of FN whereas TGF-â upregulated expression of FN in the model. Our present results indicate that the effect of TNF-á on the FN gene was not through TGF-â. Similarly to our present observations with BPAE cell system, the phenomenon of inhibition in cell proliferation and at the same time an increase in ECM gene expression has also been observed in smooth muscle cells (Lawrence et al., 1994). Nugent and Newmann (1989), in their studies with normal rat kidney cells, also noticed that TGF-â, in a dose-dependent manner, causes an induction of extracellular matrix proteins and at the same time the inhibition of cell growth. It is possible that the stimulation of synthesis of FN and collagen by

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TGF-â, may function in a negative feedback loop regulating cell growth by an autocrine–paracrine mechanism. On the basis of present data, it will be premature to suggest the precise regulatory mechanism(s) controlling expression on ECM proteins in the model system and further studies are required in this direction.

ACKNOWLEDGEMENTS We thank Jenny Zak and Lorraine Gilmore for manuscript preparation. This work was performed at the Medical College of Ohio, Toledo, Ohio and was presented in part at the American Society for Cell Biology Meeting, Washington DC, 1995. REFERENCES A RK, S MB, 1986. Type-beta transforming growth factor in human platelets: release during platelet degranulation and action on vascular smooth muscle cells. J Cell Biol 102: 1217–1223. B A, D T, 1986. Inhibition of endothelial cell proliferation by type beta transforming growth factor: interactions with acidic and basic fibroblast growth factors. Biochem Biophys Res Commun 138: 476–482. B WM, L RA, 1974. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46: 83–88. B MG, M KM, W SP, H G, S L, W JKV, Z BL, 1993. Altered expression of transforming growth factor-á and transforming growth factor-â autocrine loops in cancer cells. Adv Mol Cell Biol 7: 35–39. C M, MC TL, C E, 1987. Transforming growth factor beta is a bifunctional regulator of replication and a collagen synthesis in osteoblast-enriched cell cultures from fetal rat bone. J Biol Chem 262: 2869–2874. C P, S N, 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162: 156–159. F A, G RH, 1987. The effect of transforming growth factor-beta on cell proliferation and collagen formation by lung fibroblasts. J Biol Chem 262: 3897–3902. F-S M, M G, B W, B P, 1986. Transforming growth factor-beta inhibits endothelial cell proliferation. Biochem Biophys Res Commun 137: 295– 302. G KW, M A, 1965. An improved diphenylamine method for estimation of deoxyribonucleic acid. Nature 206: 93. G AS, L EB, S GD, M HD, 1986. Growth factors and cancer. Cancer Res 46: 1015–1020. H RL, T DR, S SM, 1986. Inhibition of endothelial regeneration by type-beta transforming growth factor from platelets. Science 233: 1078– 1080. I RA, M J, 1986. Transforming growth factorbeta stimulates the expression of fibronectin and collagen

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and their incorporation into the cellular matrix. J Biol Chem 261: 4337–4345. I RA, M J, 1987. Cell adhesion protein receptors as targets for transforming growth factor-beta action. Cell 51: 189–195. I RA, E T, M J, 1987. Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-beta. J Biol Chem 262: 6443–6446. K Y, I H, F Y, B T, 1996. Morphological identification of and collagen synthesis by periacinar fibroblastoid cells cultured from isolated rat pancreatic acini. J Gastroenterol 31: 565–571. K N, B O, S M, G AH, 1989. Macrophage production of transforming growth factor â and fibroblast collagen synthesis in chronic pulmonary inflammation. J Exp Med 170: 727–737. K-M C, L DA, C J, J P, V P, 1985. Further study of beta-TGFs released by virally transformed and non-transformed cells. Int J Cancer 35: 553–558. L DA, P R, J P, 1984a. Conversion of a high molecular weight latent beta-TGF from chicken embryo fibroblasts into a low molecular weight active beta-TGF under acidic conditions. Biochem Biophys Res Commun 133: 1026–1034. L DA, P R, K-M C, J P, 1984b. Normal embryo fibroblasts release transforming growth factors in a latent form. J Cell Physiol 121: 184–188. L R, H DJ, S GE, 1994. Transforming growth factor beta 1 stimulates type V collagen expression in bovine vascular smooth muscle cells. J Biol Chem 269: 9603–9609.  L WJF, S PE, V J, 1989. Quantitative comparison of mRNA levels in mammalian tissues: 28S ribosomal RNA level as an accurate internal control. Nucleic Acids Res 17: 10,137–10,138. M J, 1987. The TGF-beta family of growth and differentiation factors. Cell 49: 437–438. M J, 1990. The transforming growth factor-beta family. Ann Rev Cell Biol 6: 597–641. M K,  D P, I H, H C-H, 1994. Receptors for transforming growth factor-â. Immunology 55: 181–220. N MA, N MJ, 1989. Inhibition of normal rat kidney cell growth by transforming growth factor-beta is mediated by collagen. J Biol Chem 264: 18,060–18,067. O Y, N N, F S, C S, K RH, S H, S J, 1997. TGF-â1 regulation of bone sialoprotein gene transcription. Identification of a TGF-â activation element in the rat BSP gene promoter. J Cell Biochem 65: 501–512. O GK, G AAT, Y YW-H, K A, 1988. Transforming growth factor-beta-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells. J Cell Biol 107: 771–780. R JE, G L, O Y, S SM, MP J, P A, T DR, 1987. Bonederived and recombinant transforming growth factor betas are potent inhibitors of tumor cell growth. Biochem Biophys Res Commun 148: 783–789. R AB, S MB, A RK, S JM, R NS, W LM, H UI, L LA, F V, K JH, F AS, 1986. Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and

71

stimulation of collagen formation in vitro. Proc Natl Acad Sci USA 83: 4167–4171. R PG, Y MF, F KC, R NS, K P, R AH, T JD, S MB, R AB, 1987. Osteoblasts synthesize and respond to transforming growth factor-type â (TGF-â) in vitro. J Cell Biol 105: 457–463. S EH, B P, 1991. Extracellular proteins that modulate cell-matrix interactions. SPARC, tenascin and thrombospondin. J Biol Chem 266: 14,831–14,834. S Y, R DB, 1989. Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture. J Cell Biol 109: 309–315. S RS, 1995. Identification of SPARC in the anterior lens capsule and its expression by lens epithelial cells. Exp Eye Res 61: 645–648. S RS, B RC, 1993. Endotoxin alters the expression of extracellular matrix proteins by cultured endothelial cells. Cell Mol Biol Res 39: 589–599. S RS, H TM, S LJ, 1991. Biosynthesis of small proteoglycan II (Decorin) by chondrocytes and evidence for a procore protein. J Biol Chem 266: 9231–9240. S RS, K M, B RC, 1992. In: Wegmann RJ, Wegmann MA, eds. Recent Advances in Cellular and Molecular Biology. Vol. 5 Leuven, Belgium, Peeters Press. 173–180. S RS, W LS, V G, 1997. Molecular cloning of the bovine á1 (IV) procollagen gene (Col 4A1) and use in investigating the regulation of expression of type IV procollagen by retinoic acid in bovine lens epithelial cells. Cell Biol Int 21: 501–510. S G, S R, 1996. Retinoic acid: identification of specific receptors through which it may mediate transcriptional regulation of fibronectine gene in bovine lens epithelial cells. Cell Biol Int 20: 613–619. S MB, R AB, S JH, S JM, W JM, S J, 1983. Polypeptide transforming growth factors isolated from bovine sources and used for wound healing in vivo. Science 219: 1329–1331. S MB, R AB, W LM, A RK, 1986. Transforming growth factor-beta: biological function and chemical structure. Science 233: 532–534. S MB, R AB, W LM,  C B. 1987. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol 105: 1039– 1045. T K, C R E, G GR. 1987. TGFbeta inhibition of endothelial cell proliferation: alteration of EGF binding and EGF-induced growth-regulatory (competence) gene expression. Cell 49: 415–422. T DM, L RN, 1998. An essential guide to mt DNA maintenance. Nature Genetics 18: 199–200. V J, R J, J SA, 1987. Transforming growth factor beta (TGF beta) causes a persistent increase in steady-state amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts. Biochem J 247: 597–604. W LM, S DM, M T, H CC, S MB, 1987. Distribution and modulation of the cellular receptor for transforming growth factor-beta. J Cell Biol 105: 965–975. W FR, S A, S PJ, R CS, 1989. Regulation of collagen gene expression in 3T3-L1 cells. Effects of

72

adipocyte differentiation and tumor necrosis factor alpha. Biochemistry 28: 4094–4099. W JL, K T, Z Q, O CM, A JE, B WT, S J, 1991. Regulation of transformationsensitive secreted phosphoprotein (SPPI/osteopontin) expression by transforming growth factor â. Comparisons with expression of SPARC (secreted acidic cysteine-rich protein). Biochem J 273: 523–531. Y J, E M, 1992. Decreased collagen gene expression and absence of fibrosis in thyroid hormone-induced myocardial hypertrophy. Response of cardiac fibroblasts to thyroid hormone in vitro. Circ Res 71: 831–839.

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Y J, B RC, S RS, 1995. Differential effects of tumor necrosis factor-alpha on the expression of fibronectin and collagen genes in cultured bovine endothelial cells. Cell Mol Biol Res 41: 17–28. Y M, R DJ, I MG, T H, S O, S JR, S JH, R SI, B JD 1992. Transforming growth factor-beta stimulates the expression of desmosomal proteins in bronchial epithelial cells. Am J Resp Cell Mol Biol 6: 439–445.