(Received for publication, April 5, 1995; and in revised form, September 5, 1995)
From the
In this study, we identify and characterize a novel gene, CL-20, that encodes a 17.8-kDa protein with sequence and structural similarity to the growth arrest-specific gene gas3/peripheral myelin protein gene PMP22. The CL-20 protein exhibits a 43% identity with PMP22. The positions of the four lipophilic domains and the N-glycosylation site of PMP22 are conserved in CL-20, suggesting that it also is an integral membrane glycoprotein. The CL-20 gene is located on human chromosome 12 rather than 17 and encodes a 2.8-kilobase mRNA instead of 1.7-kilobase mRNA. These observations indicate that the CL-20 gene is related to but distinct from PMP22. In contrast to PMP22, CL-20 mRNA and protein are induced during squamous differentiation of rabbit tracheal epithelial cells in vitro, and Northern blot analysis and in situ hybridization demonstrated that CL-20 mRNA is most abundant in squamous epithelia. These results indicate that the high expression of CL-20 is closely correlated with squamous differentiation. The differences in tissue-specific expression and regulation between CL-20 and PMP22 suggest different roles for these two proteins. Retinoids, which inhibit squamous differentiation, repress the induction of CL-20. The retinoic acid receptor-selective retinoid SRI-6751-84 is the most effective in suppressing CL-20, suggesting that the activation of the retinoic acid receptor signaling pathway is important in this suppression.
Squamous differentiation can be observed in many tissues including the trachea, bronchus, and skin. The lining of the trachea and bronchi normally consists of a pseudostratified columnar epithelium(1) . During vitamin A deficiency or after toxic assault or mechanical injury (2, 3, 4) , regions of the mucociliary epithelium are replaced by a stratified squamous epithelium. Tracheobronchial epithelial cells also undergo squamous differentiation when cultured in medium deficient in retinoids(5, 6, 7) . In many respects, squamous differentiation in the tracheobronchial epithelial cells resembles differentiation in epidermal keratinocytes and other squamous differentiating tissues(8) . The histologically distinct layers constituting the squamous epithelium (8) exhibit distinctive patterns of expression of specific genes and are evidence that squamous differentiation is a multistage process(5, 9, 10) . Early in this differentiation process, cells irreversibly lose their proliferative potential and down-regulate the expression of the cell cycle-associated genes cdc2 and E2F-1(11, 12) . This is followed by the expression of squamous-specific genes such as transglutaminase type I(7, 13, 14, 15) , the cross-linked envelope precursors involucrin, loricrin, and cornifin(16, 17, 18, 19) , cholesterol sulfotransferase(20) , and specific keratins(9, 21) . These genes have been cloned and are shown to be regulated by a variety of agents including retinoids, which suppress the expression of squamous-specific genes(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) .
In this study, we describe the isolation and characterization of a
cDNA CL-20 that was isolated from a cDNA library prepared from
poly(A) RNA of squamous-differentiated rabbit tracheal
epithelial (RbTE) (
)cells. The open reading frame (ORF) of
CL-20 exhibits substantial sequence similarity to three cDNA sequences,
Gas3(23) , SR13(24) , and PMP22 (25, 26, 27) encoding the mouse, rat, and
human peripheral myelin protein, respectively. The peripheral myelin
protein is highly expressed in peripheral nerves (25, 26) and is induced in growth-arrested Balb/c3T3
cells(23) . Duplication and mutations in the PMP22 gene have been found to underlie several inherited diseases of the
peripheral nervous
system(28, 29, 30, 31, 32, 33) .
Although CL-20 exhibits sequence and structural similarities with
PMP22, we show that its chromosomal localization, tissue-specific
expression, and regulation are very different. Northern blot and in
situ hybridization analyses show that high CL-20 expression
correlates with squamous differentiation in vitro and in
vivo. The latter was supported by observations showing that
retinoids, which are potent inhibitors of the expression of several
squamous-specific genes, also repress the induction of CL-20.
Figure 1:
Cloning and sequence
analysis of CL-20. A, schematic comparison of pTG15 and pCL-20
cDNAs. pTG15 consists of chimeric RNA. Solid bar, CL-20
sequence; shaded bar, chimeric DNA unrelated to CL-20.
Restriction sites are indicated. TG indicates region of
homology to the degenerate oligonucleotides. Bars between dashed
lines indicate probes described in text. ORF indicates open
reading frame in pCL-20. B, induction of CL-20 mRNA expression
in relation to squamous differentiation. Total RNA (30 µg/lane)
from undifferentiated, exponentially growing and
squamous-differentiated, confluent RbTE cells was isolated and analyzed
by Northern blotting using a P-labeled full-length CL-20
probe.
Figure 4:
Human chromosomal localization of CL-20. A, Southern analysis of TaqI-digested
DNA from a panel of hybrid human/hamster or human/mouse cell lines with
the P-labeled 2.8-kb CL-20 probe. Open arrows indicate hamster-specific DNA fragments hybridizing to CL-20
probe; solid arrows indicate human-specific DNA fragments
hybridizing to CL-20 probe. Arrow heads indicate
mouse-specific DNA fragments. B, somatic cell hybrid panel
specifying the human chromosome(s) present in the hybrid cell lines. + indicates >30% of cells contain the given human
chromosome;
indicates 5-30% of cells contain given human
chromosome; D indicates the presence of multiple deletions in
the respective human chromosome. Shaded columns indicate the
two hybrid cell lines, 683 and 756, exhibiting a human hybridization
pattern with the CL-20 probe in Southern analysis. Shaded row indicates human chromosome 12 that contains the CL-20 gene.
Figure 2: Nucleotide and deduced amino acid sequence of CL-20. The deduced protein sequence is noted below the nucleotide sequence, and the translational start and stop codons are shown in bold. The length of the primer extension product is shown by the dots on the first line and ``&cjs1219;,'' which mark the ends of the primer extension product. The AUUUA instability motifs and polyadenylation signal (TATAAA) most likely used during translation are indicated in bold and underscored. The shaded amino acid sequence indicates the peptide used to raise antibodies.
Figure 3: Comparison of the amino acid sequences and structures of CL-20 and PMP22. A, comparison of the amino acid sequence of CL-20, hPMP22, and mGAS3. Two potential N-glycosylation sites at amino acids 35 and 43 were found. The second glycosylation site is conserved between CL-20 and PMP22/gas3 and is indicated by an arrow. Vertical lines indicate identical amino acids, and colons and periods indicate highly and moderately conserved changes in amino acids, respectively. The four hydrophobic regions are indicated in boldface type. For mGAS3, only the amino acids that differ from hPMP22 are indicated. B, hydrophilicity profile of CL-20. Four putative transmembrane regions can be identified.
In addition to the amino acid sequence homology, CL-20 exhibits structural similarities with PMP22. Examination of the Kyte-Doolittle hydrophilicity profile indicates that like PMP22, CL-20 contains four hydrophobic domains (Fig. 3B), suggesting that CL-20 encodes an integral membrane protein. In particular, the second hydrophobic domain (amino acids 64 through 89) is highly homologous between CL-20 and PMP22 (Fig. 3A) with 18 of the 26 amino acids being identical. CL-20 contained two potential N-glycosylation sites, amino acids 35 and 43, the latter being conserved between the two proteins (Fig. 3A).
Figure 5:
Tissue-specific expression of CL-20 mRNA.
RNA from different rabbit tissues was isolated and analyzed by Northern
blot analysis with the P-labeled 2.8-kb CL-20 probe. A, 4-h exposure; B, 8-day
exposure.
Figure 6:
Localization of CL-20 mRNA expression in
rabbit esophagus and tongue. Sections of the rabbit esophagus (A and B) and the tip of the tongue (C and D) were examined by in situ hybridization analysis
with S-labeled sense (A and C) and
antisense (B and D) CL-20
riboprobes.
Figure 7:
Comparison of CL-20 and PMP22 expression
in peripheral nerve and Balb/c3T3 fibroblasts. A, total RNA
(25 µg) from rabbit sciatic nerve were examined by Northern blot
analysis. B, poly(A) RNA (3 µg) from
Balb/c3T3 fibroblasts growing in the exponential phase (1) and
Balb/c 3T3 cells growth arrested by either confluence (2) or
serum deprivation (3) were examined by Northern blot analysis.
The blots were hybridized to
P-labeled probes for CL-20 or
mPMP22. The 2.8-kb mRNA for CL-20 and the 1.8-kb mRNA for PMP22 are
indicated.
Figure 8:
Repression of CL-20 mRNA expression by
retinoic acid and RAR- and RXR-selective retinoids. A, RbTE
cells were treated with retinoic acid (RA), the RAR-selective
retinoid SRI-6751-84 (RAR), the
RXR-selective retinoid SR11217 (RXR
),
or carrier (diff) for five days at the concentrations
indicated. Total RNA was isolated and analyzed by Northern blot
analysis using
P-labeled probes for CL-20 and
glyceraldehyde-3-phosphate dehydrogenase (GPDH). B,
dose response of CL-20 expression to the RAR-selective retinoid. A
phosphorImager (Molecular Dynamics) was used to analyze the Northern
blot in A and a second set of samples from an independent
experiment. The data for CL-20 were normalized to
glyceraldehyde-3-phosphate dehydrogenase, expressed relative to the
differentiated control(s) on each blot, and then
averaged.
To study the regulation of CL-20 at the protein level, an antiserum was raised against a peptide (CL-20-PEP3) near the amino terminus of CL-20 (Fig. 2). The antiserum recognized a protein with an apparent molecular mass of 20 kDa, which is close to the predicted molecular mass of 17.8 kDa of the unglycosylated CL-20 (Fig. 9). The synthesis of this protein was greatly increased during differentiation of RbTE cells in culture and inhibited by the presence of retinoids. The RAR-selective retinoid was just as effective in reducing the level of CL-20 protein as the expression of CL-20 mRNA. The reactivity of this antiserum with this protein was specific since the binding was abolished by the presence of homologous peptide, and no reactivity was observed with preimmune serum (not shown). The antiserum was unable to immunoprecipitate the CL-20 protein.
Figure 9:
Induction of CL-20 protein during squamous
differentiation of RbTE cells and its suppression by the RAR-selective
retinoid. Total protein extracts isolated from undifferentiated cells (lane 1), confluent squamous-differentiated cells (lane
2), and confluent cells treated with 10 (lane 3), 10
(lane 4),
10
(lane 5), 10
(lane 6), and 10
M (lane
7) RAR-selective retinoid SRI-6751-84 were analyzed by
immunoblot analysis using a rabbit antiserum against
CL-20-PEP3.
CL-20 represents a novel gene related to PMP22(22, 23, 24) . The similarity
between the two proteins is both at the level of the amino acid
sequence as well as the structure. The amino acid sequence of CL-20
exhibits a 65% similarity and a 43% identity with PMP22. The sequence
of peripheral myelin protein is highly (about 86%) conserved across
species. The homology of CL-20 with PMP22 is much smaller, suggesting
that it represents a novel gene, related to but distinct from PMP22. In
addition to their amino acid sequence homology, CL-20 and PMP22 show
structural similarities. Like PMP22, CL-20 contains four hydrophobic
domains. Likely, these genes encode membrane
proteins(23, 33) . Recently, a structural model of
PMP22 has been proposed in which the four hydrophobic domains span the
membrane either as a -sheet or as an
-helix(33) .
CL-20 may have a similar structure. The second hydrophobic region of
CL-20 and PMP22 is the most highly conserved (69% identity) and may
point at a functionally important domain that is shared between these
two proteins.
CL-20 contains two potential N-glycosylation
sites, one of which is conserved in PMP22. Although the exact
carbohydrate structure has not yet been established, human PMP22 binds
the monoclonal antibody HNK-1, which recognizes specific epitope(s)
composed of sulfated
carbohydrates(43, 44, 45) . Preliminary
observations have indicated that CL-20 may also bind HNK-1. ()These epitopes have been shown to be commonly present on
several proteins that play a role in cell adhesion and/or cell:cell
interactions such as the neural adhesion molecule (N-CAM), L1, J1,
myelin associated glycoprotein, and
P0(43, 44, 45, 46, 47, 48) .
HNK-1 antibodies have been reported to be able to inhibit aggregation
and cell adhesion, indicating that this carbohydrate is functionally
important(47, 48, 49, 50, 51) .
Another feature shared by CL-20 and PMP22 is the presence of a relatively long 3`-untranslated region containing several AUUUA instability motifs. A modulatory role in mRNA degradation under specific physiological conditions has been suggested for this motif (52) . CL-20 and the gas genes may be differentially regulated with regard to mRNA stability between cell types and during differentiation or growth arrest. Interestingly, regulation of gas3 expression during growth arrest of 3T3 cells is largely through an increase in the stability of its mRNA(28) . However, possible involvement of these instability elements in the regulation of CL-20 has yet to be determined.
Polyadenylation of CL-20 mRNA appears to
be signalled by a non-consensus motif. The region within 30 base pairs
upstream of the poly(A) sequence in the cDNA contains
three sequences (AATATA, TATAAA, and AATAGA) with one base mismatch to
the consensus polyadenylation signal sequence, which is normally highly
conserved(52) . TATAAA has been observed previously to function
as a polyadenylation signal in hepatitis B viruses(53) . In
that system, efficient use of the TATAAA signal for polyadenylation is
conferred by upstream elements. Both the non-consensus polyadenylation
signal and the presence of the AUUUA motifs hint at
post-transcriptional controls being involved, under some conditions, in
the regulation of CL-20.
Comparison of the regulation of the CL-20 and PMP22 genes indicates a number of differences. In contrast to PMP22, the expression of CL-20 is closely associated with squamous differentiation. This is indicated by the relatively high level of CL-20 mRNA expression in squamous tissues in Northern blot analysis. Moreover, in situ hybridization studies showed that in the squamous epithelium of the esophagus and tongue, CL-20 mRNA is expressed at very low levels in the basal layer containing undifferentiated cells but becomes highly expressed in the differentiated, suprabasal layers. The association of CL-20 expression with squamous differentiation is confirmed in cultured RbTE cells in which CL-20 mRNA and protein are present at low levels in undifferentiated cultures and are dramatically induced in squamous-differentiated cells. No expression of PMP22 could be detected in squamous-differentiated cells. On the other hand, expression of PMP22 is particularly high in Schwann cells and growth-arrested Balb/c 3T3 cells(23) . Both PMP22 and CL-20 are expressed in peripheral nerve, but while the former is down-regulated upon injury and up-regulated upon regeneration(24, 29, 54) , the latter is not. Likewise, while PMP22 is greatly induced when Balb/c 3T3 cells become growth arrested (23) , CL-20 mRNA is expressed at low levels, and its expression is not significantly altered during growth arrest.
Retinoids are important regulators of differentiation in
tracheobronchial epithelial cells(5) . They promote mucociliary
differentiation and are potent inhibitors of squamous differentiation.
The latter is indicated by the suppression of the induction of several
squamous-specific genes such as transglutaminase type I and cornifin (18, 55, 56) . In this study, we demonstrate
that retinoids suppress the expression of CL-20. This inhibition is
observed both at the protein and mRNA levels. It is likely that many of
the effects of retinoids on gene expression in tracheobronchial
epithelial cells are mediated either directly or indirectly by the
nuclear retinoid receptors RAR and RXR(57) . The RAR-selective
retinoid was very potent in suppressing CL-20, suggesting that the
activation of the RAR-signaling pathway is important in this
suppression. Preliminary data indicated that CL-20 mRNA is extremely
stable in squamous-differentiated RbTE cells (t > 16 h) and that retinoid treatment has no significant effect
on the half-life of this mRNA, suggesting that retinoids regulate the
expression of this RNA at the transcriptional level.
Squamous differentiation is a multi-stage process in which irreversible growth arrest occurs early and is followed by the expression of squamous cell-specific genes(5) . The induction of CL-20 expression could be related to either of these two stages of differentiation. However, retinoids, which have been shown not to block irreversible growth arrest but to suppress the expression of squamous-specific genes, repress CL-20 expression, suggesting that the function of CL-20 protein relates to the differentiated phenotype rather than to growth arrest. This conclusion appears to be supported by the in situ hybridization data showing that the induction of CL-20 occurs at the same stage of differentiation as reported for the squamous cell markers transglutaminase type I and cornifin(5, 18, 55, 56) . The induction of these genes occurs later during squamous differentiation after commitment to irreversible growth arrest.
The CL-20 and PMP22 genes map to separate human chromosomes. CL-20 is localized on chromosome 12 while PMP22 has been mapped at 17p12-13 (27, 31, 32, 33) . Genetic alterations in the expression of PMP22 have been implicated in several neuropathies specific for the peripheral nervous system. In the majority of cases of Charcot-Marie Tooth syndrome type 1A (CMT1A), the most common subtype of hereditary motor and sensory neuropathy, the gene defect is a duplication of the PMP22 gene(31) . Dejerine-Sottas syndrome, a more severe hypertrophic, demyelinating neuropathy, as well as a minority of CMT1A cases, is associated with allelic point mutations in the same gene(33) . The peripheral hypomyelination in the trembler mouse also correlates with a point mutation in PMP22(26) . It is interesting to note that many of the mutations found in PMP22 are located in the second hydrophobic domain, which exhibits the highest homology with CL-20.
In summary, CL-20 encodes a novel gene that is related to but distinct from PMP22. The gene, although detectable in several non-squamous tissues including peripheral nerve, is highly induced during squamous differentiation both in vitro and in vivo. Although the structural similarity of CL-20 and PMP22 argues for a similarity in function, the differences in tissue-specific expression and regulation suggest that their precise roles are distinct.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34200[GenBank].