(Received for publication, November 28, 1994)
From the
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.
Tenascin C (TN-C) ()is an extracellular matrix
glycoprotein composed of six similar subunits joined at their NH
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
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.
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, or HEPES, or growing the cells at
different CO
concentrations.
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 () and 6.6 (
), 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.