Differential Expression of the Fibronectin lsoform Containing the ED-B Oncofetal Domain in Normal Human Fibroblast Cell Lines Originating from Different Tissues LAURA BORSI, ENRICA BALZA, GIORGIO ALLEMANNI, Laboratory
of Cell Biology, Istituto
per la Ricerca sul Cancro, Viale Benedetto XV, 10, 16132 Genoa, Italy
651 as well as to post-translational modifications . The alternative splicing of the FN pre-mRNA is regulated in a cell, tissue, and developmentally specific manner [4, 15, 22, 25, 26, 36, 37, 44, 46, 50, 57, 60,611. Furthermore, it has been demonstrated that the splicing pattern of FN pre-mRNA is deregulated in transformed cells and in malignancies [4, 7, 8, 15, 16, 43, 47, 48, 60, 651. In fact, the FN isoforms containing the IIICS, EDA, and ED-B sequences are expressed to a greater degree in transformed human cells and in tumor tissues than in their normal counterparts. In particular, the FN isoform containing the ED-B sequence, which is undetectable in normal adult tissues with some very rare exceptions, has a much greater expression in fetal and tumor tissues as established immunoistochemically using the mAb BC-1 specific for the B-FN isoform [ 10,14,15, 26, 37, 431. Thus, we have named the ED-B sequence “oncofetal domain.” The ED-B oncofetal domain is a complete type III homology repeat composed of 91 amino acids, which is coded for by a single exon and is the most conserved FN region with 100% and 96% homology with rat and chicken FN, respectively [44, 651. This could indicate either a more recent evolution of the ED-B sequence, with less time to diverge, or a more stringent requirement due to some unknown function(s) performed by this sequence. Here we report on the expression of the B-FN mRNA evaluated through Sl nuclease protection analysis in a panel of apparently normal human cultured fibroblasts, derived from three kinds of tissue, nonfetal skin, fetal skin, and fetal lung, and in two SV-40-transformed human fibroblast cell lines. The results show that the expression of this sequence has an extreme variability depending on the developmental stage of the donor and on the tissue of origin. The results obtained studying the different FN mRNAs are consistent with those obtained studying FN as protein using two different monoclonal antibodies (mAbs): one, IST-4, specific for all the different FN isoforms and one, BC-1, specific for the B-FN isoform. The observation that the FN splicing pattern of the various fibroblast cell lines differs depending on
Fibronectin (FN) polymorphism is due both to alternative splicing of three sequences (ED-A, ED-B, and IIICS) of the primary transcript and to post-translational modifications. The FN isoform containing the ED-B sequence (B-FN), while having an extremely restricted distribution in normal adult tissues, has a high expression in fetal and tumor tissues. On a panel of nonfetal skin, fetal skin, and fetal lung fibroblast cell lines we have studied, through S 1-nuclease protection analysis, the expression of the ED-B containing FN mRNA as well as the expression of the ED-B containing FN isoform through immunoblotting and immunofluorescence techniques, using domain specific monoclonal antibodies. The results show that the expression of B-FN in the different fibroblast cell lines has an extremely great variability depending on the developmental stage of the donor and on the tissue of origin. Moreover, we found that SV-40-transformed fibroblasts present a higher expression of B-FN mRNA with respect to their normal counterparts. An increase in the relative amount of the B-FN isoform in normal human fibroblasts was also obtained by treatment with transforming growth factorP. 0 1992 Academic Press, Inc.
INTRODUCTION Fibronectins (FNs) are high-molecular-mass adhesive glycoproteins present in the extracellular matrix and in body fluids. These molecules are involved in different biological phenomena such as the establishment and maintenance of normal cell morphology, cell migration, hemostasis, thrombosis, wound healing, and oncogenie transformation (for reviews see [2, 31, 53, 621). FN polymorphism is due to alternative splicing patterns in three regions (IIICS, ED-A, and ED-B) of the single FN primary transcript (see Fig. 1) [29, 34,45,58,
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Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND LUCIANO ZARDI’
FIBRONECTIN HDMDLOOY: tYPeI typoIt typoIll
704 erur CDNA PROBE : ED-S
MDNocloNALs: hrperln DNA fibrin
cell heprrtn DNA
FIG. 1. Model of the domain structure of a human FN subunit. The IIICS, ED-A, and ED-B regions of variability due to alternative splicing of the FN pre-mRNAs are indicated. The figure also indicates the internal homologies, macromolecules interacting with the various FN domains, the possible isoforms generated by alternative splicing of the pre-mRNA, the regions where the epitopes recognized by the mAbs BC-1 and IST-4 are located, and the FN sequence covered by the probe used to study, by Sl-nuclease analysis, the splicing patterns of the ED-B exon.
the tissue of origin is in keeping with previous observations that indicate a phenotypic heterogeneity within the fibroblast population depending on the tissue of origin even though fibroblastic cells are traditionally considered a homogeneous population. This might be related to the necessity of the fibroblasts to respond appropriately to the diverse physiological situations of the various tissues [l, 17,21,30,54 and references therein, 561. Moreover, we found that the treatment of cultured normal human fibroblasts with transforming growth factor-p preferentially increases the expression of the B-FN isoform and that SV-40.transformed fibroblasts present higher relative amounts of B-FN mRNA with respect to their normal counterparts. MATERIALS
Cell cultures, transforming growth factor-j3 (TGF-/3) treatment, and immunddotting. Cultured normal human fibroblasts from nonfetal skin (GM-5757, GM-3652, GM-4390, GM-3651, GM-5659, GM-3377, GM-3440), fetal skin (GM-5386, GM-6113, GM-1603, GM-5388), and fetal lung (GM-5387, GM-6114, GM-5389) were obtained from NIGMS Human Genetic Mutant Cell Repository (Camden, NJ), as was the SV-40-transformed cell line AG-280. The human fetal lung fibroblast cell lines WI-38, IMR-90, and MRC-5, as well as the SV-40. transformed WI-38VA13 cell line, were purchased from the American Tissue Type Culture Collection (Rockville, MD). The nonfetal skin cell lines came from subjects aged between 14 months and 25 years; the fetal skin cell lines and the fetal lung cell lines were from 13. to 20-week-old fetuses. The fetal fibroblast cell lines GM-5386 and GM5387 were from the skin and the lung of the same subject as were the cell lines GM-5388, GM-5389, GM-6113, and GM-6114. Cells were
grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal calf serum (FCS) (Northumbria Biologicals Ltd., Cramlington, UK). For TGF-6 treatment, cells were grown to confluence and treated for 3 days with 0.3% FCS medium and then for 24 h with FCS-free medium containing450pMTGF-fl (R&D System, Minneapolis, MN), while the controls were treated only with FCS-free medium [3, 91. For immunoblotting experiments cells were grown for 6 days in 3.5mm wells in 3.0 ml of medium. Media were then collected from each well, and 330 ~1 of a 10X sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 100 mM TrisHCl, pH6.8, 10% SDS, 20% 6-mercaptoethanol, 50% glycerol) was added. To estimate the total level of FN in the cultures (FN in medium + FN in the extracellular matrix), cultures were solubilized by directly adding 330 pl of the 10X sample buffer for SDS-PAGE. Samples prepared in this way were directly analyzed on a 4-18% SDSPAGE gradient followed by immunoblotting. All experiments were carried out in triplicate. SDS-PAGE and immunoblotting were performed as previously described . The preparation and characterization of mAbs IST-4 and BC-1, specific for all FN isoforms (IST-4) and for the B-FN isoform (BC-l), respectively, have been previously reported [15, 55, 641. Immunofluorescent experiments were carried out as previously described . Sl -nuclease protection analysis. Total RNA was extracted according to Chirgwin et al.  from the confluent fibroblast cell lines at passages 15 to 25. At least two different RNA extractions were made on each cell line. RNA Sl-nuclease protection analysis for the determination of the B-FN mRNA was carried out as described by Borsi et al. . The 821-base double-stranded cDNA probe for the ED-B region was prepared and labeled as previously reported  (see Figs. 1 and 2). For each RNA preparation, at least three independent Sl nuclease protection analyses were carried out. The Sl-nuclease-resistant fragments were analyzed on a 6% polyacrylamide gel containing 8 M urea, followed by autoradiography and double-dimension analysis of the resulting film by an LKB Ultrascan XL laser densitometer .
ED-B + 704
FIG. 2. On the left a schematic representation depicts the probe used to study, by Sl-nuclease analysis, the splicing pattern of the ED-B exon (hatched area). The wavy line corresponds to the sequence derived from the cloning vector (3’ end). The asterix marks the 5’ “P-labeled end. The probe fragments protected by the FN mRNA species containing (ED-B+) and not containing (ED-B-) the ED-B exon are also shown. The autoradiogram on the right shows Sl nuclease experiments using RNA from GM-5386 fetal skin fibroblasts (lane 2), GM-5387 fetal lung fibroblasts (lane 3), and GM-5659 nonfetal skin fibroblasts (lane 4). Sl-nuclease analysis of RNA from TGF-P-treated (lane 6) anduntreated (lane 5) MRC-5 fetal lung fibroblasts is also shown. Lane 1, undigested probe.
lung of the same fetuses. In all three cases we observed that the percentage of B-FN mRNA in lung fibroblasts was 2.5 times higher than that observed in skin fibroblasts (Fig. 4). Similar results were obtained using Northern blot and dot blot analyses with appropriate cDNA probes covering the ED-B sequence or sequences common to all FN mRNAs (data not shown). We have also tested whether the level of the ED-B containing mRNA in a cell line changed depending on the number of passages. We found that it was not significantly affected within the range between 10 and 30 passages. Furthermore, by immunoblotting using two mAbs, one specific for all FN isoforms (IST-4) and the other specific only for the B-FN isoform (BC-1) (see Fig. l), we have compared the amount of total FN and of B-FN isoform in total extracts and in the conditioned media of all the above reported cell lines. The results were in line with the data obtained studying the mRNAs (Fig. 5 and data not shown). Figure 5 shows immunoblots of the conditioned culture media of three nonfetal skin fibroblast cell lines and of three fetal lung fibroblast cell lines obtained using the mAbs IST-4 and BC-1. While in the
Expression of the B-FN Isoform in Different Human Fibroblast Cell Lines
We have used Sl-nuclease analysis to evaluate the relative amount of B-FN mRNA with respect to total FN mRNAs in 7 nonfetal skin, 4 fetal skin, and 6 fetal lung fibroblast cell lines. In Fig. 2 we have depicted a schematic representation of the cDNA probe used to study the splicing pattern of the ED-B exon as well as the probe fragments protected by FN mRNA species containing or not containing the ED-B sequence. Figure 2 also shows autoradiograms of gels of probe fragments obtained after different Sl-nuclease protection experiments. We found extensive variability in the percentage of the B-FN mRNA among the 17 normal fibroblast cell lines tested. In fact, the values ranged from 0.5% (GM5757 nonfetal skin fibroblasts) to 30% (WI-38 fetal lung fibroblasts). The lowest values were seen in fibroblast lines derived from nonfetal skin, while the highest in lines derived from fetal lung (see Fig. 3). Furthermore, among the fetal fibroblast lines we observed that those deriving from skin showed lower relative amounts of B-FN mRNA with respect to those deriving from lung (Fig. 3). To eliminate differences due to the developmental stage or to genetic heterogeneity, we have analyzed three pairs of cell lines deriving from the skin and the
Non fetal skin fibroblasts (7 cell lines)
Fetal skin fibroblasts
(4 ccl) lines)
Fetal lung fibroblasts (6 cell lines)
FIG. 3. Relative abundance of the B-FN mRNA, determined by Sl-nuclease analysis, in nonfetal skin fibroblasts, fetal skin fibroblasts, and fetal lung fibroblasts. Each cell line is represented as a grey circle. These results, obtained by densitometric scanning of autoradiograms deriving from Sl-nuclease experiments similar to those shown in Fig. 2 represent the average of six independent experiments on two different RNA preparations in which the values differed by no more than 20%.
GM-6113 GM-61 14 GM-5388 GM-5389
FIG. 4. Diagram of the relative abundance of B-FN mRNA in three pairs of skin (white columns) and lung (hatched columns) fibroblast lines derived from the same fetuses. The results obtained by laser densitometric scanning of autoradiograms deriving from Sl-nuclease experiments similar to those shown in Fig. 2 represent the average of six independent experiments on two different RNA preparations differing by no more than 20%.
conditioned media of nonfetal skin fibroblasts the B-FN was undetectable, it was clearly visible in the conditioned media of fetal lung fibroblasts even though all these cell lines release similar amounts of total FN into the media as evaluated using the mAb IST-4 (Fig. 5). Immunofluorescence experiments were also carried out on all 17 fibroblast cell lines using the mAbs IST-4 (which recognizes all FN isoforms) and BC-1 (which recognizes only the B-FN isoform). Results in this case were also in line with both the data obtained studying the mRNAs and those obtained in immunoblotting experiments. In fact, while all the different cell cultures presented a similar positive reaction using the mAb IST-4 (total FN), using the mAb BC-1 (B-FN) no staining was visible in the extracellular matrix of the nonfetal cells, a barely detectable staining was present in the matrix of the fetal skin fibroblasts, and a more intense staining was visible in the matrix of the fetal lung cell cultures (Fig. 6).
ent 48 h after TGF-fi withdrawal from medium (data not shown). Figure 2 shows an autoradiogram of an Sl-nuclease analysis of RNA from the MRC-5 cell line before (lane 5) and after (lane 6) TGF-P treatment. TGF-P induced higher increases of the relative amount of B-FN mRNA in fibroblast lines which, in standard cultured conditions, showed lower levels of B-FN mRNA than in fibroblast lines with higher basal levels of B-FN mRNA (data not shown). We also tested the percentage of B-FN mRNA in WI38VA13 and AG-280 cell lines, which are the SV-40transformed counterparts of WI-38 and IMR-90 fetal lung fibroblast cell lines, respectively. In both cases we observed a two-to-threefold higher relative amount of B-FN mRNA in the SV-40-transformed cells compared to that of the untransformed counterparts (Fig. 7). In particular, in the WI-38VA13 line, more than 90% of the FN mRNA molecules contained the ED-B sequence. In fact, this cell line has been shown to be the best source for the purification of the B-FN isoform [lo]. DISCUSSION
We have studied the expression of the FN isoform containing the ED-B oncofetal domain in different lines of normal human fibroblasts. We have observed that while in some cell lines this FN isoform may represent 30% of the total FN produced, in others the B-FN isoform represents only 0.5%. These differences in the rela-
Expression of the B-FN Isoform in TGF-P-Treated and SV-40-Transformed Human Fibroblast Cell Lines As previously reported, TGF-P increases the relative amount of the B-FN  and B-FN mRNA . Here we have extended the experiments to a larger number of fibroblast cell lines that produce, in standard culture conditions (see Material and Methods), different levels of B-FN. In all the lines tested we observed a two- to fivefold increase of the relative amount of the B-FN mRNA after TGF-P treatment. This effect was still pres-
IST-4 FIG. 5.
Immunoblotting experiments of conditioned culture media of the nonfetal skin fihroblast lines GM-3652 (lane l), GM-5757 (lane 2), and GM-4390 (lane 3), and the fetal lung fibroblast lines MRC-5 (lane 4), IMR-90 (lane 5), and WI-38 (lane 6), using the mAb IST-4, which recognizes all the different FN isoforms (left) and the mAb BC-1 which recognizes only the B-FN isoform (right). While all the six cell lines release similar amounts of total FN into culture medium, as established using the mAb IST-4, only the fetal lung fibroblast cell lines release detectable amounts of the B-FN isoform with ED-B specific BC-1 mAb into medium.
FIG. 6. Indirect immunofluorescence experiments using the mAb BC-1, which recognizes only the B-FN isoform, and IST-4, which recognizes all different FN isoforms on (from top to bottom) GM-3652 nonfetal skin fibroblasts, GM-5388 fetal skin fibroblasts, and GM-5389 fetal lung fibroblasts. Only fetal fibroblast cell lines show detectable amount of the B-FN isoform.
tive amounts of B-FN produced by different normal human fibroblast cell lines were observed both by studying the mRNA through Sl nuclease analysis and by studying the FN protein using domain specific mAbs. Furthermore, we observed that fetal-derived human fibroblasts showed higher relative amounts of B-FN than human nonfetal fibroblasts. Among the human fetal fibroblast cell lines, we observed an expression of the B-FN iso-
form higher in lung-derived than that in skin-derived lines. This difference was also confirmed in pairs of fibroblast cell lines deriving from the lung and the skin of the same fetuses, respectively, thus excluding that this difference may be due to the developmental stage, genetic heterogeneity, or unknown peculiarities of the donor. The observations that (1) cultures of fibroblasts de-
t g’ 2 z s 8 m
FIG. 7. Cpmparison of the relative abundance of B-FN mRNA, determined by Sl-nuclease analysis, in the normal fetal lung fibroblasts WI-38 and IMR-90 (white columns) and in their SV-40-transformed counterparts WI-38VAl3 and AG-280 (hatched columns). The results, obtained by densitometric scanning of autoradiograms similar to that shown on the right, represent the average of six independent Sl-nuclease experiments on two different RNA preparations in which the values differed by no more than 20%. The autoradiogram on the right shows Sl-nuclease experiments using RNA from the cell line WI-38 and its SV-lo-transformed counterpart WI-38VA13. The probe fragments protected by FN mRNA species containing (ED-B+) and not containing (ED-B-) the ED-B exon are also indicated.
rived from fetal tissues express more B-FN than those derived from nonfetal tissues and (2) among fetal skin and fetal lung fibroblast lines the latter have the higher production of B-FN are in line with in uiuo studies. In fact, it has been previously demonstrated that fetal tissues express higher amounts of B-FN compared to adult tissues [15, 22, 26, 37, 44, 48, 501. Furthermore, in our previous immunohistochemical studies, we either did not find or found only very small amounts of B-FN in fetal skin while we observed higher amounts of this FN isoform in fetal lung . The observation that fibroblasts have different FN splicing patterns depending on the tissue of origin is in line with previous studies reporting that normal fibroblasts from different tissues are differently specialized in order to appropriately respond to the diverse physiological or pathological situations of various tissues. Indeed, for instance, cultured fibroblasts synthesize different types of collagen, hormone receptors, or specific enzymes depending on their anatomical site of origin [ 1,54,59]. Furthermore, it has been shown that fetal and lung fibroblast lines derived from the same fetus differ in replication rate, cell numbers at confluence, and cell volume . There have also
been observed differences in in vitro growth potential of human skin fibroblast lines derived from papillary or reticula dermis . We have previously reported [15, 651 that the fetal lung fibroblast cell line WI-38 produces very low amounts of B-FN, while it is greatly expressed in the SV-40-transformed WI-38VA13 cell line. However, in the present study we have demonstrated that all the fetal lung cell lines tested produce high amounts of BFN. The discrepancies with our previous data may be due to the fact that in our earlier experiments, the WI38 cell line was obtained indirectly from the American Tissue Culture Collection (ATCC) following passages through different laboratories. Thus, we have repeated the comparison by Sl-nuclease analysis of the levels of FN mRNA containing the ED-B sequence in two pairs of normal and SV-40-transformed human fibroblast cell lines obtained directly and recently from ATCC. The results clearly show that, at least in these two cases, there is a two- to threefold higher relative amount of the B-FN mRNA in the SV-40-transformed lines compared to their normal counterparts. This has also been operationally confirmed, since a SV-40-transformed cell line has been shown to be the best source for the purification of the B-FN isoform [lo]. These data disagree with those reported by Schwarzbauer et al.  and Norton and Hynes  who did not observe any significant increase in the relative amount of B-FN mRNA in transformed embryonic rat and chicken cells compared to their respective normal counterparts. This discrepancy may be due either to the different cellular systems used or to the fact that the Sl-nuclease experiments reported by these authors were difficult to evaluate from a quantitative point of view [44,58]. However, independently of the in vitro results, there is general agreement on the fact that the stroma of normal adult tissues does not show, with some rare exceptions, significant amounts of B-FN as either mRNA or protein, while it is present in large amounts in the stroma of fetal tissues and of malignant neoplasia [14, 15, 22, 26, 37, 43, 44, 48, 50, 651. Another oncofetal FN isoform recognized by the mAb FDC-6, has been described . The epitope recognized by this mAb is localized within the IIICS sequence (see Fig. 1) and is composed of an oligosaccharide linked to a hexapeptide by 0-glycosylation . Thus, the oncofetal epitope recognized by the mAb FDC-6 originates from an altered glycosylation of FN in fetal tissues and tumors and it is not related to the ED-B sequence. Loridon-Rosa et al.  reported that this oncofetal FN isoform, similar to the B-FN isoform, is undetectable in normal breast and in benign breast tumors while it is present in 60% of invasive breast carcinomas. These observations have suggested that normal stroma1 fibroblasts are induced by factors released by tumor cells to produce these oncofetal FN isoforms. The possi-
bility of such a paracrine effect by which tumor cells modulate the extracellular components of the stroma has already been demonstrated in the case of the neoexpression of tenascin in the stroma of breast carcinoma  and is strongly suggested by neoexpression of proteolytic enzymes or extracellular matrix components in tumor stroma [5, 20, 241. This hypothesis is also supported by the appearance of new antigens on the surface of stromal fibroblasts of carcinoma  and by the distribution of the B-FN isoform only in a thin layer of stroma which surrounds tumor cell nests in in situ breast carcinoma (L. Zardi et al., in preparation). Chiquet-Ehrismann et al.  have shown that the factor responsible for the neoexpression of TN in the stroma of breast carcinomas is TGF-fi which is released by neoplastic cells. We have previously demonstrated the ability of TGF-P to increase the relative amount of B-FN in cultured human cells [3,9]. Here we have extended our studies to a larger panel of cultured fibroblast lines and have confirmed these results. Other molecules have been shown to be able to modulate FN splicing in cell cultures. Burton-Wurster et al.  reported that dibutyryl cyclic AMP decreases expression of ED-A containing FN in cultured canine chondrocytes while Bennet et al.  reported that retinoic acid dramatically increases the expression of ED-A and ED-B FN isoforms in cultured chicken chondrocytes. An increased expression of the FN isoform containing the IIICS sequence, which also undergoes alternative splicing, has recently been reported in cultured endothelial cells after TGF-P treatment . This recent observation gives functional significance to the regulatory effects of TGF/3 on splicing of pre-mRNA since it is known that this alternatively spliced portion of the FN molecule, IIICS, is involved in modulating cell attachment, migration, and spreading [28, 32, 33,421. These data suggest that TGF-fl may control th extracellular matrix composition and function, not only by increasing the accumulation of FN and other extracellular matrix proteins [3a and references therein], but also by modifying the relative amount of the different FN isoforms. This is achieved through the modulation of the splicing of FN premRNA, and possibly pre-mRNA of other extracellular matrix proteins as well, with a consequent heightened expression of isoforms which may have specific biological functions. Alternative RNA splicing is an important widespread mechanism of gene regulation. The differential expression of exons into mature RNA is often under developmental and/or tissue specific regulation. It has been demonstrated that alternative splicing is regulated by information encoded in the gene transcript (cis) but also requires diffusible nuclear factor(s) (truns), which in turn are regulated by extracellular environmental factors such as cytokines, endotoxins, growth factors, and hormones [ll, 12, 27, 38, 491. Surprisingly, studies on
the effects of such environmental stimuli on splicing patterns of messenger RNA precursors are extremely rare . Thus, this may be a fertile future research area together with the study of the biological function(s) of the sequences which are variably expressed both in normal and in pathological conditions. This study has been partially supported by AIRC funds. We thank Miss Antonella Gessaga for skillful secretarial assistance and Mr. Thomas Wiley for manuscript revision. We are indebted to Prof. L. Santi for his support and encouragement. We are grateful to Dr. B. Azzarone and to Dr. F. E. Baralle for helpful discussions.
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