(Received for publication, August 26, 1994; and in revised form, November 8, 1994)
From the
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 1 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
1 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.
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), ()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
1 (TGF-
1),
dibutyryl cyclic adenosine monophosphate ((Bu)
cAMP), and
fucoidan on fibronectin synthesis and the alternative splicing of the
ED-A and ED-B segments of fibronectin pre-mRNA.
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.
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.
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.
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.
Figure 4:
Effects of TGF-1, (Bu)
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
TGF
1-treated cultures, n = 2; for (Bu)
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- 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-
1, p = 0.041; 30% by
fucoidan, p = 0.043) and had little effect on the
alternative splicing of ED-B. (Bu)
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-1, (Bu)
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 TGF
1) or three
((Bu)
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).
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.()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- 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-
on fibronectin synthesis by
articular chondrocytes in cartilage explant has been observed
(Burton-Wurster and Lust, 1990). TGF-
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-
(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-
1 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-
1.
The addition of (Bu) 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)
cAMP. The down-regulation of
ED-B(+) fibronectin does not agree very well with the presumed
role of (Bu)
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-1 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.
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].