©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Alternative Splicing Pattern of the Tenascin-C Pre-mRNA Is Controlled by the Extracellular pH (*)

(Received for publication, November 28, 1994)

Laura Borsi Enrica Balza Barbara Gaggero Giorgio Allemanni Luciano Zardi (§)

From the Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Viale Benedetto XV, 10, 16132 Genoa, Italy

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Alternative splicing of primary transcripts is an ubiquitous and reversible mechanism for the generation of multiple protein isoforms from single genes. Here we report that in cultured normal human fibroblasts, small pH variations of the culture medium (from 7.2 to 6.9) strikingly modify the alternative splicing pattern of the tenascin-C primary transcript. Since such extracellular pH variations occur in many normal and pathological conditions, microenvironmental pH may be an important element for the regulation of RNA alternative splicing in vivo.


INTRODUCTION

Tenascin C (TN-C) (^1)is an extracellular matrix glycoprotein composed of six similar subunits joined at their NH(2) terminus by disulfide bonds(1, 2, 3, 4) . During development, TN-C displays a time- and a space-dependent tissue distribution with morphogenically significant boundaries. Each human TN-C subunit includes three types of structural modules: 14.5 epidermal growth factor-like repeats, 16 type III homology repeats, and a COOH-terminal knob made up of a sequence with homology to the globular domain of the beta and chains of human fibrinogen (Fig. 1A). TN-C is coded for by a single gene, and its expression is regulated by a single promoter. Structurally and functionally different human TN-C isoforms are generated by the alternative splicing of the TN-C transcript, eight type III repeats being included or omitted in the mRNA (Fig. 1A)(5, 6, 7) . In cultured normal human fibroblasts, alternative splicing generates two main TN-C mRNAs of 8 and 6 kb, which are easily distinguished by Northern analysis(8) .


Figure 1: Effect of environmental pH on the relative steady-state levels of the two TN-C mRNAs in cultured human fibroblasts. A, model of the domain structure of a human TN-C subunit. The fibronectin (FN)-like repeats undergoing alternative splicing are indicated in gray. TN-C isoforms that are generated by the 6- and 8-kb TN-C mRNAs are also shown. B, Northern analysis of poly(A) RNA from human cultured lung fibroblasts (WI-38) using the probe HT-11 (see ``Materials and Methods''). At confluence (8 days after plating), the medium was replaced with fresh medium supplemented with 20% FCS, at different pH. Poly(A) RNA was extracted after 48 h of incubation. Lane C, poly(A) RNA from cell cultures in which the medium was not changed. C, Northern analysis of poly(A) RNA from skin cultured fibroblasts (GM-5757) using the probe HT-11 (see ``Materials and Methods''). To confluent cultures (8 days after plating), the medium was replaced with fresh medium supplemented with 3 mg/ml bovine serum albumin at different pH. Poly(A) RNA was extracted after 96 h of incubation. Lane C, poly(A) RNA from cultures in which the medium was not changed.



Alternative pre-mRNA splicing is an important reversible mechanism of gene regulation, which enables a single gene to encode multiple functionally different proteins(9, 10, 11, 12, 13) . Although it is generally held that the pattern of alternative RNA splicing is only cell type-specific or developmentally regulated, its reversibility suggests that it might be tuned by extracellular cues, which enable cells to generate proteins with activities adequate to respond to mutated environmental conditions. Here we report that in cultured normal human fibroblasts, small pH variations of the culture medium (from 7.2 to 6.9) strikingly modify the alternative splicing pattern of the human TN-C primary transcript. Since such extracellular pH variations occur in many normal and pathological conditions, microenvironmental pH may be important for the regulation of RNA alternative splicing in vivo.


MATERIALS AND METHODS

Cells

Lung (GM-5387, GM-6114, WI-38, IMR-90, and MRC-5) and skin fibroblasts (GM-5757, GM-3440, GM-6113, and GM-5386) were obtained from NIGMS, Human Genetic Mutant Cell Repository (Camden, NJ). All the cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc., Paisley, UK) supplemented with 20% fetal calf serum (FCS, Boehringer Mannheim) in a 5% CO(2) atmosphere. Cells were utilized at passages 15-24, at least 7 days after seeding.

In all the experiments described here, the different pH of the medium was obtained using different concentrations of sodium bicarbonate (from 5.0 to 60 mM). Identical results were obtained also modifying medium pH by HCl, NaOH, NH(3), or HEPES, or growing the cells at different CO(2) concentrations.

RNA Purification and Northern Analysis

Total RNA and poly(A) RNA were isolated from cultured cells using the procedure reported by Borsi et al.(14) . Northern blot analyses of 2 µg of poly(A) RNA were carried out using a human TN-C cDNA probe (HT-11) covering a DNA sequence common to all TN molecules(14) . A human glyceraldehyde-3-phosphate dehydrogenase cDNA probe (Clontech Laboratories, Palo Alto, CA) was used to normalize Northern blots. The cDNA probes were P-labeled using a random primed DNA labeling kit (Boehringer Mannheim). Mono- and bidimensional analyses of the autoradiographic films (Trimax-XD, 3M, Savona, Italy) were carried out using an LKB Ultroscan XL laser densitometer (Pharmacia/LKB, Uppsala, Sweden).


RESULTS AND DISCUSSION

Cultured human skin and lung fibroblasts, at confluence, showed very different steady-state levels of the two TN-C mRNAs and corresponding proteins. In fact, while skin fibroblasts showed almost exclusively the 8-kb mRNA, lung fibroblasts accumulated mainly the 6-kb mRNA (Fig. 1, B and C, lanes C). However, lung fibroblasts have a much higher ability to acidify the medium with respect to skin fibroblasts, since they produce much higher quantities of lactate(15) . Indeed, 7-8 days after plating, skin fibroblasts induce minimal pH variations in the culture medium while lung fibroblasts induce significant acidification. Identical results were obtained using the different cell lines indicated under ``Materials and Methods.''

Substitution of the media of confluent cultures of both lung and skin fibroblasts with media at different pH, followed by Northern analysis of the TN-C mRNAs, demonstrated that the environmental pH controls the steady-state levels of the two TN-C mRNAs. At a pH slightly above 7.0, fibroblasts from both skin and lung preferentially accumulate the 8-kb TN-C mRNA, while at pH below 7.0, they preferentially accumulate the 6-kb TN-C mRNA, irrespective of the chemicals used to modify the pH of the media. The presence or absence of 20% FCS or cytokines did not modify this behavior (Fig. 1, B and C).

These results may be due either to modification of the RNA alternative splicing pattern or to a different instability of the two TN-C mRNAs in cells cultured in media with different pH. The results of the experiments shown in Fig. 2A demonstrated that the two TN-C mRNAs have equal instability at either pH 7.6 or 6.6.


Figure 2: Effect of environmental pH on the relative instability and amounts of the two TN-C mRNAs. A, equal instability of the two TN-C mRNAs in human fibroblasts cultured in media having pH 7.6 (box) and 6.6 (bullet), respectively. To lung fibroblasts, the culture medium was replaced 5 days after plating with medium having the pH indicated above and 5 µg/ml actinomycin D (ACT-D, Sigma) were added. The relative amounts of the two TN-C mRNAs were established by Northern analysis after different incubation times. B, Northern analysis of poly(A) RNA from lung and skin fibroblasts using the HT-11 and the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA probes (see ``Materials and Methods''). To confluent cultures (8 days after plating), the medium was replaced with fresh DMEM at different pH supplemented with 20% FCS in the case of lung fibroblasts and 3 mg/ml bovine serum albumin in the case of skin fibroblasts. Poly(A) RNA was extracted from the cultures after 2 days (lung) and 4 days (skin) of incubation. C, amounts of the two TN-C mRNAs in human skin fibroblasts incubated (8 days after plating) for 4 days with fresh DMEM at different pH, supplemented with 3 mg/ml bovine serum albumin. The amounts of the TN-C mRNAs (expressed in arbitrary units) were evaluated through densitometric scanning of films derived from Northern blot analyses using the HT-11 probe and normalized with the glyceraldehyde-3-phosphate dehydrogenase probe. Bar = standard error.



In skin fibroblasts kept in FCS-free media at pH 7.4 for 4 days, the 6-kb TN-C mRNA represented about 2% of the total mRNA, while at pH 6.7 it amounted to 98%. However, the total amount of TN-C mRNA (6 + 8 kb) did not show significant changes (Fig. 2, B and C). This finding rules out the hypothesis that the observed variation in the alternative splicing pattern may be due to an increase of the total amount of the TN-C primary transcript, with a possible saturation of some limiting factor(s) involved in the regulation of alternative splicing. In these experiments cells were incubated in FCS-free medium to isolate the effect of environmental pH from the effects of cytokines that are known to stimulate TN synthesis(16) .

The present findings demonstrate that RNA alternative splicing may be considered not only cell type-specific or developmentally regulated but also, at least in the case of TN-C, controlled by microenvironmental pH variations. Considering that similar pH variations occur in vivo in many normal and pathological conditions and that it is very unlikely that such a control mechanism is evolved only for the TN-C transcript, environmental pH may be important for the in vivo regulation of RNA alternative splicing. This notion assumes even greater importance in light of the recent observation that organs in the human body are not homogeneous in terms of pH and that specific organ systems maintain a pH that is significantly different from the systemic pH(17) .

Several reports have shown that the large TN-C isoform is expressed at the onset of important cellular processes that entail active cell migration, proliferation or tissue remodeling such as in neoplasia, in wound healing, and during development(8, 14, 18, 19, 20, 21, 22) . These observations suggest that the extracellular pH, regulating the expression of the different TN-C isoforms, may be important in the regulation of cellular migration and proliferation.

The intracellular events leading to pH-dependent alternative RNA splicing are for the moment only a matter of speculation. However, it is well established that modification of the microenvironmental pH induces, in normal cells, parallel modification of the steady-state intracellular pH(23, 24, 25) ; as a consequence, such modification could affect the activity of a number of enzymes that play a role in the regulation of RNA alternative splicing and, likewise, the affinity for RNA and protein sequences of regulatory elements.

The modulation of the alternative splicing of different primary transcripts induced by extracellular pH, as well as the molecular events which lead to such regulation, are presently under investigation.


FOOTNOTES

*
This work was supported in part by Associazione Italiana per la Ricerca sul Cancro and ``Progetto Finalizzato: Applicazioni Cliniche della Ricerca Oncologica'' of the Consiglio Nazionale delle Ricerche. 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.

§
To whom correspondence should be addressed. Tel.: 39-10-3534901; Fax: 39-10-352855.

(^1)
The abbreviations used are: TN-C, tenascin C; kb, kilobase(s); DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum.


ACKNOWLEDGEMENTS

We thank Thomas Wiley for manuscript revision.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.