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(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) (
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)
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
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 (
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.
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 6243-6245
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)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) .
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.
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 atmosphere. Cells were utilized at passages
15-24, at least 7 days after seeding.
, or HEPES, or growing the cells at
different CO 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).
) 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.
)
We thank Thomas Wiley for manuscript revision.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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