©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alternative Splicing of ED-A and ED-B Sequences of Fibronectin Pre-mRNA Differs in Chondrocytes from Different Cartilaginous Tissues and Can Be Modulated by Biological Factors (*)

(Received for publication, August 26, 1994; and in revised form, November 8, 1994)

Dai-wei Zhang (§) Nancy Burton-Wurster (¶) George Lust

From the James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The alternative splicing of the ED-A and ED-B segments of fibronectin pre-mRNA was examined in epiphyseal, costal, and meniscal cartilage from 3-week-old beagles and in nasal, tracheal, articular, and meniscal cartilage from 1- and 2-year-old Labrador retrievers. In contrast to the 100% expression of ED-B(+) mRNA that has been reported for embryonic chick cartilage (Bennett, V. D., Pallante, K. M., and Adams, S. K.(1991) J. Biol. Chem. 266, 5918-5924), all cartilages studied expressed both the ED-B(+) and ED-B(-) forms of fibronectin mRNA with the exception of the trachea, in which expression was 100% ED-B(-). Of all cartilages studied, only the meniscus had detectable levels of ED-A(+) mRNA. Placing articular cartilage chondrocytes in primary monolayer culture dramatically up-regulated the expression of ED-A(+) mRNA to 25% of the total, and this expression was further increased by the addition of transforming growth factor beta1 or fucoidan to the culture medium. The expression of ED-B(+) mRNA remained at about 18% in the cultured chondrocytes and was not further affected by either transforming growth factor beta1 or fucoidan. In contrast, dibutyryl cyclic adenosine monophosphate decreased the relative expression of both the ED-A(+) and ED-B(+) forms of fibronectin pre-mRNA. We concluded that the expression of ED-B(+) fibronectin remains relatively high in chondrocytes from cartilaginous canine tissues (15-35%) with the exception of the trachea, in contrast to the less than 10% expression of ED-B(+) fibronectin reported for other non-fetal tissues.


INTRODUCTION

Fibronectin is an important glycoprotein in extracellular matrices and body fluids and belongs to the category of adhesive molecules, which bridge the interactions between cells and the matrix (see Hynes, 1990).

A single gene encodes the fibronectin molecule, which is composed of three types of homologous repeating units (types I, II, and III). The alternative splicing of the primary transcript (pre-mRNA) of the fibronectin gene generates different fibronectin isoforms, which are distinguished from each other by the inclusion or exclusion of certain regions of the molecule (Hynes, 1985). Three such alternatively spliced regions, extra domains ED-A and ED-B and the IIICS region, have been identified (Vibe-Pedersen et al., 1984; Odermatt et al., 1985; Schwarzbauer et al., 1987). While the IIICS region can be spliced partially, ED-A and ED-B are spliced in or out completely. The presence of ED-A or ED-B distinguishes the cellular form of fibronectin from the plasma fibronectin synthesized by liver in which both ED-A and ED-B are excluded. The alternative splicing of fibronectin pre-mRNA was found to be tissue specific and development dependent (Hynes, 1990; Huh and Hynes, 1993). A study screening the alternative splicing of fibronectin among different adult tissues showed that the fibronectin mRNAs from those adult tissues was ED-A(+) at a range from 0 to 25% and ED-B(+) from 0 to 10% (Magnuson et al., 1991). A developmental change in the alternative splicing pattern of the fibronectin pre-mRNA has also been observed in chick chondrogenesis (Bennett et al., 1991). It was found that in the chick embryo, mesenchymal cells produce ED-B(+)/ED-A(+) fibronectin mRNA. After differentiation, at chick development stage 27, the splicing pattern is switched from ED-B(+)/ED-A(+) in the mesenchymal cells to ED-B(+)/ED-A(-) in embryonic chondrocytes and ED-B(-)/ED-A(+) in embryonic muscle cells.

The presence of fibronectin has been demonstrated in adult canine articular cartilage at the protein level (Wurster and Lust, 1982; Burton-Wurster et al., 1988). With the use of a monoclonal antibody to ED-A, it was found that less than 2% of canine articular cartilage fibronectin contains ED-A (Burton-Wurster and Lust, 1990). In a previous preliminary study using the polymerase chain reaction (PCR), (^1)we provided direct evidence that fibronectin mRNA is expressed in adult canine articular cartilage and that, in contrast to chick embryo cartilage, both the ED-B(+) and ED-B(-) forms of fibronectin are expressed (Zhang et al., 1993). In this study, the expression of ED-B(+) fibronectin mRNA as well as ED-A(+) fibronectin mRNA was quantitated in articular cartilage and in several other cartilaginous tissues using a ribonuclease protection assay. Also, in an attempt to understand the regulation of fibronectin synthesis and, more importantly, the alternative splicing of fibronectin pre-mRNA, a primary chondrocyte culture system was used to study the effects of transforming growth factor beta1 (TGF-beta1), dibutyryl cyclic adenosine monophosphate ((Bu)(2) cAMP), and fucoidan on fibronectin synthesis and the alternative splicing of the ED-A and ED-B segments of fibronectin pre-mRNA.


EXPERIMENTAL PROCEDURES

Cartilage Tissues

The epiphyseal cartilage, costal cartilage, and meniscal cartilage were taken from three 3-week-old beagles at necropsy. Specifically, epiphyseal cartilage was taken from the humeral head and the femoral condyles, and meniscus was taken from the knee joints of the young beagles. Nasal cartilage, tracheal cartilage, articular cartilage (pooled from several synovial joints), and the meniscus were taken from two adult Labrador retrievers (1.5 years old each). Additional articular cartilage was obtained from a 12-month-old Labrador retriever.

Chondrocyte Cultures

Chondrocytes were isolated and primary cultures were established in monolayer by seeding at a cell density of 10^6 in 10 ml of Ham's F12 medium containing 10% fetal bovine serum (Leipold et al., 1992). When the cells reached confluence, the medium was replaced with 10 ml of fresh complete medium along with different additions for the treatments. Recombinant human TGF-beta1 (R& Systems Inc., Minneapolis, MN) was added at a concentration of 10 ng/ml, (Bu)(2) cAMP (Sigma) was added at a concentration of 0.5 mM, and fucoidan (Sigma) was added at a concentration of 100 µg/ml. Each treatment, as well as the control, was incubated in duplicate or triplicate. The cells were treated for 72 h under those conditions. The medium was then replaced with 5 ml of fresh complete medium with the different additions. L-[S]Methionine (1200 Ci/mmol, Amersham Corp.) was added into each flask at a final concentration of 10 µCi/ml. The cells were then pulse labeled for 6 h. At the end of the labeling, the medium was collected and stored at -20 °C until used. RNA was isolated from the cells as described below.

Determination of Total Protein Synthesis and Fibronectin Synthesis

Total protein synthesis was analyzed by perchloric acid precipitation of the S-labeled proteins in the culture medium. Fibronectin was purified from media by affinity chromatography with a gelatinSepharose column as described (Engvall and Ruoslahti, 1977; Pena et al., 1980). Aliquots of the fractions of urea eluate were counted with a liquid scintillation counter. The presence of fucoidan did not interfere with the purification of fibronectin.

RNA Extraction

From Cultured Chondrocytes

When the cell culture was completed, the medium was removed, and the flask was washed twice with phosphate-buffered saline. Total cellular RNA was isolated from primary chondrocytes by modification of a method described by Nemeth et al.(1989).

From Cartilage Tissue

Cartilages were taken from the stated sources, quick-frozen in liquid nitrogen immediately after the isolation, and then stored at -70 °C until use. RNA was isolated from cartilage tissues with a protocol developed by Adams et al.(1992). In this method, the lysis mixture was extracted once with an equal volume of water (diethyl pyrocarbonate treated)-saturated phenol and once with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1). This was followed by ultracentrifugation through a 2-ml cesium trifluoric acid cushion (d = 1.5) at 35,000 rpm for 18 h at 18 °C. This modification achieved good purification of the RNA from contaminating proteoglycans.

Reverse Transcription-PCR

Two primers spanning the ED-A region of the canine fibronectin gene were derived from a human fibronectin sequence (Magnuson et al., 1991). Primer 1 (the antisense primer located in the type III repeat 12) was 5`-AGAGCATAGACACTCACTTC-3`, and primer 2 (the sense primer located in the type III repeat 11) was 5`-CAGAAATGACTATTGAAGGC-3`. Cellular RNA (0.5 µg) from untreated confluent chondrocytes was used to reverse transcribe the first strand cDNA. The reverse transcription was made in the following 20-µl reaction solution: 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 8 mM MgCl(2), 1 mM each dNTP, 50 pmol of the antisense primer, 50 units of Moloney murine leukemia virus reverse transcriptase, 20 units of RNasin® (a ribonuclease inhibitor), and 5 mM dithiothreitol. The reaction was incubated at 42 °C for 45 min, and the reverse transcriptase was inactivated by heating at 95 °C for 5 min at the end of the reaction. The reverse transcription mixture was then diluted to 100 µl with 1 times PCR buffer (50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl(2)). 50 pmol of second primer and 2.5 units of Taq polymerase were added, and PCR was performed for 30 cycles using the following parameters: initial denaturing, 94 °C for 1.5 min; amplification cycle (30 cycles), annealing (55 °C for 1 min), extension (72 °C for 1 min, 50 s), denaturing (94 °C for 20 s); termination, annealing (55 °C for 1 min) followed by extension (72 °C for 7 min).

ED-B cDNA was obtained in the same way using one primer located in the type III repeat 7 (sense primer, 5`-GTTATAGAATTACCACAACC-3`) and another primer located within the ED-B region (antisense primer, 5`-CCAGCCCTGTGACTGTGTAG-3`). A longer ED-B cDNA, which contains the whole length of the canine fibronectin ED-B, was obtained with the same sense primer but a different antisense primer located in the type III repeat 8 (5`-GGGATCCTTGGTGAATCGCAGGTCAG-3`).

The amplified DNA was analyzed by electrophoresis on agarose gels. The gel bands corresponding to ED-A or ED-B inclusions were cut out of the gel and purified with the Prep-a-gene kit from BIO-RAD and subcloned into plasmid vectors as described below.

Cloning of Canine ED-A and ED-B cDNA

A T-A cloning scheme (Marchuk et al., 1990) was used to clone canine fibronectin ED-A and ED-B. For the ED-A clone, PCR II, a T-vector purchased from Invitrogen was used. The ED-A cDNA amplified by PCR as described above was ligated with the vector in the conditions specified by the manufacturer. Transformation was performed, and positive clones were selected by PCR using the two ED-A primers. The resultant clone was termed PCRII-WA and contained the complete ED-A sequence. For ED-B clones, a T-vector made of pGEM-3Z from Promega was used. The pGEM-3Z-based T-vector was prepared using a method developed by Marchuk et al.(1990). Briefly, 2 µg of the pGEM-3Z plasmid was digested with a blunt end restriction enzyme SmaI and then incubated with the Taq polymerase at 72 °C for 2 h in the presence of only dTTP. The reaction mixture was extracted once with phenol/chloroform, precipitated with ethanol, and dissolved in sterile water. Ligation to the amplified ED-B cDNAs, transformation, and screening were performed in the same way as for the amplified ED-A cDNA. The resultant clones were termed pGEM-EB, containing the partial ED-B sequence, and pGEM-WB, containing the whole length of the ED-B sequence.

Sequencing of the ED-A and ED-B Clones

The PCRII-WA clone containing the whole length of canine ED-A sequence and the pGEM-WB clone containing the whole length of canine ED-B sequence were sequenced from both ends of the canine cDNA inserts in the plasmids. Plasmid DNA was prepared using a QIAGEN mini-prep kit. The DNA was dissolved in water at a final concentration of 1 µg/µl and sequenced with the Applied Biosystems Institute (Foster City, CA) sequencing system.

RNase Protection Assay

Preparation of DNA Templates for the Labeling of RNA Probes

To obtain the ED-A probe, the ED-A-containing clone, PCRII-WA, was digested with RsaI to linearize the plasmid. The digestion generated a DNA template that included 65 bp of the ED-A sequence, 227 bp of the flanking type III repeat 12 sequence, and the T7 promoter of the vector. The digestion mixture was then extracted once with phenol/chloroform and loaded on to a 2% NuSieve-agarose gel. The gel was stained with 1 µg/ml ethidium bromide. The appropriate band was cut out and purified with a Prep-a-gene kit from BIO-RAD. This probe was termed PCRII-EA. The DNA template for the ED-B riboprobe was generated similarly with the exception that EcoRI was used to linearize the plasmid pGEM-EB. The probe contained 169 bp of the canine ED-B sequence and 190 bp of the flanking type III repeat 7 sequence and the SP6 promoter. A schematic of the probes is presented in Fig. 1.


Figure 1: Schematic of the alternatively spliced mRNA structure of fibronectin ED-A and ED-B region. The mRNAs are represented by boxes. The alternatively spliced regions are indicated by boxes with slashedlines. The solidlines above the mRNA boxes represent the cDNA templates for RNA probes used in the RNase protection assay.



RNA Probe Labeling

An in vitro transcription kit from Promega was used to prepare the riboprobe for the RNase protection assay. The reaction mixture included 20 units of SP6 (for ED-B) or T7 (for ED-A) RNA polymerase and 50 µCi of [P]CTP. Incubation was at 38 °C for 60 min, followed by DNase digestion, extraction with phenol/chloroform, and precipitation of the RNA at -70 °C in ammonium acetate/ethanol according to the manufacturer's instructions. The RNA pellet was dissolved in hybridization buffer B (Ambion, Austin, TX).

RNase Protection Assay

An RPA II kit from Ambion was used for the RNase protection assay. Cellular RNA (5 µg) was precipitated with 4 M sodium acetate (1/10 volume) and ethanol (2.5 volumes) at -70 °C for 15 min. The RNA pellet was dissolved in 20 µl of hybridization buffer B containing 5 times 10^4 cpm of RNA probe. The hybridization mix was heated to denature the RNA, hybridization was permitted to continue overnight at 42 °C, and single stranded RNA was then digested with RNase (RNase® A/T1) at 33 °C for 60 min. At the end of the digestion, the RNA was precipitated, and the RNA pellet was resuspended in 5 µl of the RNA loading buffer, heated at 85 °C for 5 min, and then analyzed on a 5% polyacrylamide, 7 M urea gel. After electrophoresis, the gel was transferred to a piece of chromatography paper, dried under vacuum, and exposed to x-ray film at -70 °C with an intensifying screen.

Quantitation of the mRNA

The relative radioactivity of the protected fragments was quantitated by scanning the autoradigraph with an LKB densitometer (LKB Inc., Bromma, Sweden). The data was collected and analyzed with computer software (GSXL) provided by LKB Inc. The relative density of each band was calculated and corrected by the sizes of the relevant bands.

Statistical Analysis

A Student's t test was used to compare the results of the effects of TGF-beta1, dibutyryl cyclic AMP, and fucoidan on fibronectin synthesis and on the alternative splicing of the ED-A and ED-B regions of fibronectin pre-mRNA.


RESULTS

Canine Fibronectin ED-A and ED-B Sequences Are Highly Homologous to Other Mammalian Species

Canine fibronectin cDNAs were amplified successfully with the reverse transcription-PCR approach using cellular RNA from cultured chondrocytes as described under ``Experimental Procedures.'' Both canine ED-A and ED-B fibronectin cDNAs were generated in this manner with primers derived from the human fibronectin sequence. The identity of the amplified cDNAs was confirmed by characteristic restriction enzyme digestion and DNA sequencing of the PCR products (data not shown). The PCR-amplified canine fibronectin cDNAs with inclusion of ED-A or ED-B were subsequently cloned into bacterial plasmid vectors and used as templates for the preparation of riboprobes in the RNase protection assay.

Sequencing analysis of the canine fibronectin ED-A and ED-B segments showed that these sequences are very homologous to the human and rat sequences (Fig. 2, A and B). As for the ED-A sequence, on the nucleic acid level, there are 2 nucleotide differences between canine and human ED-A (99% homology) and 13 nucleotide differences between canine and rat ED-A (95% homology); on the protein level, the amino acid sequences of ED-A are identical between canine and human (100% homology), but there are 5 amino acid differences between canine and rat ED-A (94% homology). As for the ED-B sequence, on the nucleic acid level, there are 6 nucleotide differences between canine and human ED-B (97% homology) and 9 nucleotide differences between canine and rat ED-B (96% homology); on the protein level, the sequences in all three species are identical.



Figure 2: A, canine fibronectin ED-A cDNA sequence; B, canine fibronectin ED-B cDNA sequence. Nucleotides that differ between the rat and the canine sequences and between the human and the canine sequences are indicated.



Alternative Splicing of ED-A and ED-B of Fibronectin Pre-mRNA in Different Cartilaginous Tissues

The ribonuclease protection assay was used to quantitatively determine the ratio of ED-B(+) and ED-B(-) fibronectin mRNA in articular cartilage. Other cartilaginous tissues were also examined. Trachea was different in that its fibronectin mRNA had neither ED-A nor ED-B (A-B-). In this respect, the fibronectin expressed in the trachea would resemble plasma fibronectin, which is essentially A-B-. ED-A was excluded in the fibronectin mRNA from all of the cartilages except for menisci (Fig. 3A). The meniscus from young dogs expressed 15.3 ± 4.0% (n = 3) ED-A(+) fibronectin mRNA, while only trace amounts of ED-A(+) fibronectin was identified in the meniscus in adult dogs (<3%). Fig. 3presents a representative ribonuclease assay for each tissue. Table 1presents the average of all data obtained. ED-B(+) fibronectin mRNA was expressed in all the cartilage tissues except for the trachea and was between 13-35% of the total fibronectin mRNA in those cartilages (Table 1). The variation observed is very consistent with variation observed on the protein level in a previous study (Burton-Wurster et al., 1989).


Figure 3: Alternative splicing of ED-A and ED-B of fibronectin pre-mRNA in different cartilage tissues. Total cellular RNA was extracted from a variety of cartilaginous tissues of both young and old dogs as described under ``Experimental Procedures.'' RNA was allowed to hybridize with the P-labeled riboprobes covering a portion of the ED-B or ED-A segments. After digestion with RNase, the protected fragments were separated by electrophoresis. The gel was dried and exposed to x-ray film, and the radioactivity of the protected fragments were quantitated. See ``Experimental Procedures'' for details. A, the riboprobe used covered a portion of the ED-A segment (see Fig. 1, schematic); B, the riboprobe used covered a portion of the ED-B segment (see Fig. 1, schematic). The cartilaginous source of the RNA and the age of the dog from which it was obtained (young (3 weeks) indicated as y; adult (1.5 years) indicated as a) was as follows: lane1, tRNA (a); lane2, epiphyseal (y); lane3, costal (y); lane4, meniscal (y); lane5, meniscal (a); lane6, nasal (a); lane7, tracheal (a); lane8, articular (a); lane9, probe.





Effects of TGF-beta1, (Bu)(2)cAMP, and Fucoidan on Fibronectin Synthesis and the Alternative Splicing of ED-A and ED-B of Fibronectin Pre-mRNA in Cultured Chondrocytes

Isolated, primary chondrocytes were cultured to confluence in monolayers, treated with either TGF-beta1, dibutyryl cyclic AMP, or fucoidan, a polysulfated fucose polymer from marine algae, for 3 days. Newly synthesized fibronectin in the medium was determined and expressed as the percentage of the total new protein in the medium. Previous experiments suggest that over 90% of the newly synthesized fibronectin in control and (Bu)(2) cAMP-treated cultures will be present in the culture medium (Leipold et al., 1992). TGF-beta 1 showed stimulatory effects on the synthesis of fibronectin in primary chondrocyte cultures. Compared with the control, the addition of TGF-beta1 increased fibronectin synthesis relative to total protein synthesis by 41% (p = 0.099). On the contrary, (Bu)(2) cAMP had a moderate inhibitory effect (35% decrease, p = 0.082), and fucoidan decreased the relative fibronectin synthesis dramatically by 80% (p < 0.05) (Fig. 4).


Figure 4: Effects of TGF-beta1, (Bu)(2) cAMP, and fucoidan on fibronectin synthesis in articular chondrocytes. Chondrocytes were isolated and cultured as described under ``Experimental Procedures.'' S-Labeled fibronectin and total protein were quantitated by enzyme-linked immunosorbent assay and precipitation with perchloric acid, respectively, as described in the text. For control and TGFbeta1-treated cultures, n = 2; for (Bu)(2) cAMP and fucoidan-treated cultures, n = 3. The relative synthesis of fibronectin with respect to total protein synthesis was significantly decreased in those cultures treated with fucoidan (p < 0.05).



Although TGF-beta and fucoidan had opposite effects on fibronectin synthesis in the primary chondrocytes, they behaved similarly in regulating the alternative splicing of ED-A and ED-B of fibronectin pre-mRNA, i.e. they both increased the inclusion of ED-A (37% by TGF-beta1, p = 0.041; 30% by fucoidan, p = 0.043) and had little effect on the alternative splicing of ED-B. (Bu)(2) cAMP decreased the inclusion of both ED-A (by 55%, p = 0.017) and ED-B (by 45%, p = 0.052) in fibronectin mRNA (Fig. 5).


Figure 5: Effects of TGF-beta1, (Bu)(2) cAMP, and fucoidan on the alternative splicing of the ED-A and ED-B segments of fibronectin pre-MRNA in articular chondrocytes. The experiment was performed as described for Fig. 4except that total mRNA was extracted and purified as described under ``Experimental Procedures.'' The results of a ribonuclease protection assay similar to the ones depicted in Fig. 3were quantitated, and the results are depicted graphically. Results are an average of data obtained from two (control and TGFbeta1) or three ((Bu)(2) cAMP and fucoidan) incubation flasks. * and** indicate a significant shift in the alternative splicing pattern with respect to the alternate splicing pattern of ED-A or ED-B, respectively, in the control (p < 0.05).




DISCUSSION

It has been observed that the fibronectin sequence is highly conserved among mammalian species (Hynes, 1990). Sequence analysis of the two canine fibronectin cDNA clones described here provided evidence that, as in other mammalian species, the ED-A and ED-B fibronectin sequence is highly conserved. Although the exact functions of the fibronectin ED-A and ED-B domain are still not well elucidated, the fact that they are highly conserved among species suggests that they might have important functions in the extracellular matrix.

The alternative splicing of fibronectin pre-mRNA has been found to be development dependent and tissue specific (Hynes, 1990). For example, the alternative splicing of fibronectin pre-mRNA switches from A+B+ to A-B+ in chick chondrogenesis (Bennett et al., 1991). In late developmental stages of the chick embryo, the cartilage fibronectin is almost exclusively 100% ED-B(+) and ED-A(-). In a previous study, we estimated that at the protein level, human articular cartilage contained detectable but variable (up to 30% or more) amounts of ED-B(+) fibronectin. Less than 2% of ED-A(+) fibronectin was detected at the protein level in canine articular cartilage (Burton-Wurster et al., 1988, 1989). The alternative splicing patterns of fibronectin pre-mRNA in nonfetal cartilage were unknown. As shown in this study, ED-B was included in 23 ± 8% of the total fibronectin mRNA from canine articular cartilage, but no ED-A mRNA was detected. In contrast, as reported by Magnuson et al.(1991), most adult tissues express only low levels (ranging from 0 to 10%) of ED-B(+) fibronectin mRNA. It should be pointed out that the studies reported herein were done with the ribonuclease protection assay while those of Magnuson et al.(1991) relied on PCR technology, which we have found to be less quantitative. If the studies of Magnuson et al.(1991) are correct, however, it appears that cartilage expresses the highest levels of ED-B(+) fibronectin among the adult tissues examined so far. Results of Rencic et al.(^2)in human articular cartilage show an inclusion rate for ED-B of 30% and thus are consistent with our results.

One possibility for the high levels of ED-B(+) fibronectin in adult articular cartilage is that this splicing pattern is chondrocyte specific. It could also be reasoned that this type of splicing pattern is related to the weight-bearing and weight-transmitting function of the articular cartilage or that it is associated with the presence of type II collagen, which is the predominant type of collagen in articular cartilage. Our data showed that the alternative splicing of ED-A and ED-B differs in different cartilaginous tissues. In other words, the inclusion of ED-B in fibronectin mRNA is not chondrocyte specific since the chondrocytes in trachea expressed virtually no ED-B(+) fibronectin. Neither is weight bearing associated with the inclusion of the ED-B segment in fibronectin mRNA since nasal cartilage, which has little weight-bearing function, expressed (on average) similar levels of ED-B(+) fibronectin as articular cartilage. Meniscus, which contains both type I and type II collagen, expressed comparable levels of ED-B(+) fibronectin to articular cartilage, which contains type II but no type I collagen. On the other hand, trachea, which contains type II collagen, expressed no ED-B(+) fibronectin. This rules out any strict association between the presence of type II collagen and ED-B(+) fibronectin. Thus, we still have no clue as to the function of ED-B(+) fibronectin.

Chondrocytes are the only type of cells present in mature articular cartilage. The relatively small number of cells in the cartilage tissue in vivo necessitates that a large amount of tissue be available to isolate sufficient RNA for the ribonuclease protection assay. Therefore, we turned to a cell culture system, as opposed to an explant system, to study the gene expression of the fibronectin in response to various factors. The more abundant mRNA is easier to detect in such a system. To minimize the potential dedifferentiation of chondrocytes in cell culture, primary chondrocytes were used in these studies and were isolated and cultured as previously described (Leipold et al., 1992). All factors were added after the cells became confluent; thus, effects of the factors on proliferation should be minimal, and cell numbers should be comparable for all treatments. As anticipated from our previous study (Burton-Wurster et al., 1988; Leipold et al., 1992) at the protein level, the expression of ED-A(+) fibronectin mRNA was up-regulated to 25% in chondrocytes in primary culture. The expression of ED-B(+) fibronectin mRNA by cultured chondrocytes was 18%, not very different from the 22 ± 8% found in articular cartilage. In contrast, Bennett et al.(1991) demonstrated a decline from 100% in vivo to almost 0% in embryonic chick chondrocytes in monolayer culture. The discrepancy might be due to the differences in the source of the chondrocytes and in the culture conditions.

TGF-beta is known to have regulatory effects on cell growth and extracellular matrix formation (Ellingsworth et al., 1986; Morales and Roberts, 1988; Redini et al., 1988). The stimulatory effect of TGF-beta on fibronectin synthesis by articular chondrocytes in cartilage explant has been observed (Burton-Wurster and Lust, 1990). TGF-beta also has regulatory effects on the alternative splicing pattern of fibronectin pre-mRNA. The expression of both ED-A(+) and ED-B(+) fibronectin was found up-regulated in cultured fibroblasts in the presence of TGF-beta (Borsi et al., 1990). In this study, we observed the up-regulation of the expression of ED-A(+) fibronectin (by 37%) in cultured primary chondrocytes, although the expression of ED-B(+) fibronectin was not changed significantly. In a preliminary study, TGF-beta1 had little effect on the alternative splicing of ED-A in cartilage explants. Since chondrocytes in articular cartilage in vivo express little ED-A(+) fibronectin, one can speculate that the expression of ED-A(+) fibronectin must first be turned on before it can be modulated by TGF-beta1.

The addition of (Bu)(2) cAMP to primary chondrocyte cultures reduced the inclusion of ED-A in fibronectin by 55% on the mRNA level. This is comparable with the decrease on the protein level observed in a previous study (Leipold et al., 1992). The inclusion of ED-B in fibronectin, which was not looked at in the previous study, was also found decreased by 45% under the influence of (Bu)(2) cAMP. The down-regulation of ED-B(+) fibronectin does not agree very well with the presumed role of (Bu)(2) cAMP in maintaining the differentiated state of the cultured chondrocytes since, if one assumes ED-B(+) to be characteristic of the chondrocyte phenotype, an up-regulation of the ED-B(+) splice form would be expected.

Fucoidan, a sulfated fucopolysaccharide of the marine algae Fucus vesiculosus, was found to inhibit the synthesis of fibronectin by arterial smooth muscle cells (Vischer and Buddecke, 1991). We observed the same inhibitory effects in cultured primary chondrocytes. The addition of fucoidan reduced fibronectin synthesis relative to total protein synthesis by 80%. The alternative splicing of fibronectin pre-mRNA was also affected. The inclusion of ED-A was increased by 30%, but the inclusion of ED-B was not changed significantly.

In summary, the alternative splicing pattern of ED-A and ED-B in fibronectin pre-mRNA was different in chondrocytes from different cartilaginous tissues. Most noteworthy was the absence of ED-B(+) fibronectin in tracheal cartilage and the relatively high levels of ED-A(+) fibronectin in meniscal cartilage. When chondrocytes were isolated from cartilage and placed in culture, the inclusion of ED-A was up-regulated while the inclusion of ED-B was hardly affected. Addition of TGF-beta1 or fucoidan to the cultured chondrocytes, while having opposite effects on total fibronectin synthesis, further increased inclusion of ED-A but did not change the expression of ED-B(+) fibronectin. In contrast, dibutyryl cyclic AMP decreased the synthesis of fibronectin and the inclusion of both ED-A and ED-B.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AR35664 and a grant-in-aid from the Ciba-Geigy Corporation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U16207[GenBank], U16208[GenBank].

§
Submitted to the Graduate Faculty of Cornell University in partial fulfillment of the requirements for a Ph.D. degree.

To whom all correspondence should be addressed. Tel.: 607-256-5651; Fax: 607-256-5608.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; TGF-beta1, transforming growth factor beta1; (Bu)(2), dibutyryl; bp, base pairs.

(^2)
Rencic, A., Lewis, S. D., Gehris, A. L., and Bennett, V. D.,(1995) Osteoarthritis Cartilage, in press.


ACKNOWLEDGEMENTS

We thank Alma Jo Williams and Margaret Vernier-Singer for technical assistance and Dorothy Scorelle for assistance in the preparation of the manuscript.


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