(Received for publication, May 31, 1994; and in revised form, November 2, 1994)
From the
Ichthyosis vulgaris is an autosomal dominant disorder of keratinization characterized by mild hyperkeratosis and reduced or absent keratohyalin granules in the epidermis. Profilaggrin, a major component of keratohyalin granules, is reduced or absent from the skin of individuals with ichthyosis vulgaris. In this report, we have further characterized the molecular basis of low profilaggrin expression, which occurs in this disease. In situ hybridization revealed little profilaggrin mRNA in ichthyosis vulgaris-affected epidermis. In keratinocytes cultured from the epidermis of affected individuals, the abundance of profilaggrin was reduced to less than 10% of normal controls, while the mRNA level was decreased to 30-60% of controls. Expression of K1 and loricrin, other markers of epidermal differentiation, were not affected. Nuclear run-on assays indicated that the decrease in mRNA levels was not caused by aberrant transcription. Nucleotide sequencing of 5`-upstream, 3`-noncoding, and flanking regions of the profilaggrin gene from ichthyosis vulgaris-affected individuals revealed only minor changes, probably due to genetic polymorphisms. Our results indicate that defective profilaggrin expression in ichthyosis vulgaris is a result of selectively impaired posttranscriptional control.
Ichthyosis vulgaris (IV) ()is an autosomal dominant
skin disorder reported to occur in as many as 1 in 250 of the normal
population(1) . Affected skin appears scaly and is
characterized histologically by hyperkeratosis and a decreased or
absent granular layer(2) . In addition, keratohyalin granules,
an ultrastructural landmark in the granular layer, are either absent or
reduced and structurally abnormal(3) . Other clinical symptoms,
including hyperlinear palms and soles, a personal or family history of
atopy, and keratosis pilaris are often associated with IV(1) .
Although the disease is well described clinically, the etiology is
poorly understood.
Filaggrin is a cationic protein that aggregates keratin intermediate filaments in the stratum corneum of the epidermis (for review, see (4) ). Profilaggrin, the precursor of filaggrin, is first expressed in the granular layer and marks the terminal stages of epidermal differentiation. The phosphorylated profilaggrin accumulates in keratohyalin granules and later undergoes dephosphorylation and proteolysis to filaggrin. Profilaggrin and filaggrin are noticeably decreased or absent from the epidermis of patients with IV(5) . We have previously shown that keratinocytes cultured from affected individuals maintain structural and biochemical phenotypic characteristics of the disorder(6) . For example, very little profilaggrin is detectable by immunohistochemical staining on Western blots of extracts obtained from IV keratinocytes compared with controls. These data are consistent with the absence of keratohyalin in this disorder. This suggests a pronounced decrease in profilaggrin synthesis and/or accumulation as is seen in the skin biopsies from affected individuals.
Recently, the structure of the human profilaggrin gene has been reported by two different laboratories(7, 8) . The gene (see Fig. 3A) contains three exons interrupted by two introns of 9,713 and 570 bp, respectively. The 5`-noncoding region (75 bp) is divided into two exons separated by the large intron. The coding region begins in the second exon and continues in the third exon, where 10-12 highly repetitive filaggrin sequences of exactly 972 bp reside. The number of filaggrin repeats varies between individuals and is inherited in a Mendelian fashion(9) . The amino terminus of profilaggrin contains a calcium binding domain consisting of two EF-hands resembling those present in the S-100 family of proteins(7) . It has recently been shown that the S-100 domain of profilaggrin binds calcium(8, 10) . Hence, profilaggrin may not only function as a keratin aggregating protein, but it may also play a critical role in the regulation of calcium-dependent events during epidermal differentiation(7) .
Figure 3: Expression of profilaggrin mRNA in IV-affected individuals and controls. A, diagram demonstrating the regions covered by the probes (2, exon 2; 3, exon 3) used in hybridization are indicated. B, Northern analysis of total RNA from the three IV-affected individuals and unrelated controls studied in Fig. 2. The amount of RNA in each lane was normalized based on the glyceraldehyde-3-phosphate dehydrogenase level. RNA was fractionated on a 1% glyoxal gel, blotted, and hybridized with a repeat of the third exon of the profilaggrin gene (proFG) and glyceraldehyde-3-phosphate dehydrogenase. The same blot was also hybridized to keratin 1 (K1) (3`-end), human loricrin (3`-end), and HS26 probes. Much lower hybridization signal from the loricrin probe in the middle pair is unexplained; all RNAs were on the same blot. It should also be noted that the intensity of the hybridization signal generated from each probe does not reflect the relative abundance of the RNAs as the specific activities of the probes were not identical.
Figure 2: SDS-PAGE and immunoblotting analyses of profilaggrin in IV-affected individuals and controls. Equal protein loadings of extracts from IV-affected keratinocytes cultured from three unrelated individuals (IV)and normal controls (C) were analyzed by PAGE. Controls in the first two pairs were unaffected offspring, while the control in the third pair was an unrelated unaffected age- and sex-matched individual. Lane F contains foreskin epidermal extract. The proteins were blotted onto nitrocellulose paper and reacted with antibodies directed against human profilaggrin (proFG) and filaggrin (FG) (a), neutral-basic keratins (K1, K5, and K6) (b), and loricrin (LORI) (c). Identity of the 30 kDa loricrin band was confirmed in parallel studies with antiserum generously supplied by Dennis Roop (Houston, TX) (data not shown).
To investigate further the association between decreased profilaggrin expression and IV, we studied profilaggrin mRNA levels in vivo by in situ hybridization and utilized a human epidermal keratinocyte culture system as an in vitro model. We analyzed profilaggrin expression at the protein, steady-state mRNA, and transcriptional levels in keratinocytes cultured from individuals with IV as well as from appropriate, unaffected family members and age- and sex-matched normal controls. Our data indicate that selectively impaired posttranscriptional regulation results in reduced profilaggrin mRNA and protein in IV.
2) The profilaggrin 5` upstream region (885 bp) was
generated from human genomic DNA using the following
oligonucleotides ((8) , GenBank M96943):
5`-TGGTAGGAGGCACAATGT-3` and 5`-GAGCCTGCTGGGTACTGA-3`. Amplification
using PCR was performed using Taq polymerase (Promega Corp.).
Conditions for PCR were 94 °C for 1 min, 50 °C for 1 min, and
72 °C for 1 min, for 30 cycles.
3) The profilaggrin-3`-noncoding region (517 bp) was generated from human genomic DNA using an upstream oligonucleotide that included the stop codon (underlined) of human profilaggrin gene(9) : 5`-GATACTATTACTATGAGTAAGA-3` and 5`-GACATCTAATTCTGGCCATGG-3`. Amplification using PCR was performed using Taq polymerase (Promega Corp.). Conditions for PCR were 94 °C for 1 min, 42 °C for 1 min, and 72 °C for 1 min, for 30 cycles.
4) The K1 cDNA clone (a gift of Dr. D. Roop, Baylor College of Medicine, Houston, TX) is a subclone of pK456 (110 bp) and contains the BamHI-PstI fragment of pK456, which encodes the carboxyl-terminal end domain and 3`-noncoding region of the human K1 gene(19) .
5) The loricrin-specific probe (120 bp) was prepared based on the 3`-noncoding sequence of human loricrin (20) using the following oligonucleotides: 5`-GTACCACGGAGGCGAAGGAGT-3` and 5`-GGTTGGGAGGTAGTTGTACAG-3`. Conditions used for PCR were 94 °C for 1 min, 59 °C for 1 min, and 72 °C for 1 min, for 30 cycles.
6) The glyceraldehyde-3-phosphate dehydrogenase, pHcGAP plasmid was obtained from ATCC.
7) The cDNA for human S26 ribosomal protein (HS26) was a gift of Dr. P. Fort, Montpellier Cedex 2, France.
8) Genomic DNA was prepared from cultured fibroblasts of normal and IV-affected individuals as described previously(21) .
Figure 1:
In situ hybridization of skin
from IV-affected individuals and controls. Biopsies of unaffected (C) and affected (IV) individuals were hybridized
with S-labeled antisense filaggrin riboprobe. Reduced
silver grains were viewed by light microscopy. B, basal layer; S, spinous layer; G, granular layer; C,
cornified layer; Bar = 30
µm.
The basis of comparison relied on the use of glyceraldehyde-3-phosphate dehydrogenase to normalize RNA on the blots. However, recent evidence suggests that glyceraldehyde-3-phosphate dehydrogenase expression may vary as a function of proliferative state and physiological conditions within the cell(25, 26, 27) . Keratinocytes used in this study were at postconfluence, when proliferation is reduced(28) . Our assumption was that the rate of proliferation of normal and IV-affected keratinocytes is similar. However, it is possible that development, differentiation, and/or metabolic activity of keratinocytes from IV-affected individuals differ from normal. Hence, regulation of glyceraldehyde-3-phosphate dehydrogenase may be altered, and the RNA profile may be inaccurate. Therefore the same blots were hybridized with a second probe made from a cDNA for HS26 ribosomal protein RNA, which shows invariant expression among several cell lines(29) . The phosphorimaging analysis indicated that the levels of profilaggrin mRNA normalized to HS26 were 24, 67, and 40% of the corresponding controls. The result is similar to that obtained using glyceraldehyde-3-phosphate dehydrogenase as a standard for normalization, indicating profilaggrin mRNA levels in IV keratinocytes are about 45% of normal controls.
Keratinocytes
cultured from individuals with IV and unaffected controls were
harvested 3 days after reaching confluence. Harvesting was done 24 h
after feeding, which increases profilaggrin mRNA transcription. ()Nuclei and total RNA were then isolated. Hybridization of
the nascently transcribed labeled RNA to a human filaggrin repeat, K1,
and glyceraldehyde-3-phosphate dehydrogenase fragments revealed
virtually identical levels of transcription between keratinocytes from
normal and IV-affected individuals (Fig. 4). In contrast,
experiments using nuclei from subconfluent keratinocytes gave very low
signal for profilaggrin transcription (data not shown). Because the
assay utilized a probe of filaggrin repeat from exon 3, the result also
suggested that transcriptional pausing in either the first or second
exon was not a cause of reduced profilaggrin mRNA in IV-affected
individuals. In order to test more rigorously for possible
transcriptional pausing in profilaggrin expression, sarkosyl which
overcomes pausing (24) was added. It had no effect on signal
intensity in either IV-affected or normal individuals (data not shown),
suggesting that transcriptional pausing was not a factor.
Figure 4:
Nuclear run-on transcription analysis of
the profilaggrin gene in confluent keratinocytes cultured from
IV-affected individuals and normal controls. Nuclei were isolated from
control (C) and IV-affected keratinocytes (IV), and
run-on transcription assays were performed as described under
``Materials and Methods.'' The pair on the farright is from two of the individuals studied in Fig. 2. The two pairs on the left are not, but
expression of profilaggrin mRNA and protein was identical to that shown
in Fig. 2and Fig. 3. Equal counts/min of P-labeled RNA were hybridized to filters loaded with
linearized plasmids (10 mg each) containing inserts of the following:
3` terminus of K1 cDNA (K1), cDNA of a filaggrin repeat (FG), and glyceraldehyde-3-phosphate dehydrogenase. Linearized
plasmid pGEM-3 was used for an estimation of the nonspecific
hybridization signal.
The 3`-noncoding region of the profilaggrin gene was amplified from three individuals with IV, two of whom were members of the same family. The amplified 564-bp genomic fragment contained a sequence from 10 bp upstream of the stop codon to 47 bp downstream of the poly(A) addition site. Sequence analysis showed a few variations from that of normal unaffected individuals or the published sequence(9) . The stop codon and poly(A) addition site were intact and located as previously reported. No alterations that might increase the instability of the RNA were identified. We believe that these variations are due to genetic polymorphism and do not contribute to the marked decrease in profilaggrin mRNA seen in IV.
We have previously demonstrated that keratinocytes cultured from patients with IV exhibited several characteristics of the disorder including decreased expression of profilaggrin protein(6) . In the present paper, we show that profilaggrin mRNA levels are reduced in IV epidermis and in keratinocytes cultured from IV-affected individuals and that the abnormal profilaggrin expression is a specific defect and does not reflect a more general defect in expression of markers of epidermal differentiation. Our results also suggest that the defect occurs primarily at the posttranscriptional level. The supporting evidence for this conclusion is that the transcription level of the profilaggrin gene between keratinocytes cultured from IV-affected individuals and controls (as determined by run-on assays) is similar, irrespective of the levels of corresponding steady-state cellular RNA.
Decreased profilaggrin in IV is a result of deficient profilaggrin mRNA, which in turn indicates either aberrant gene transcription or posttranscriptional regulation. Northern analysis using a single filaggrin repeat as probe consistently showed that total RNA isolated from confluent IV keratinocytes contained less profilaggrin mRNA than their normal counterparts (Fig. 3B). Our in vitro study of profilaggrin and loricrin expression agrees with the in situ hybridization (Fig. 1) and antibody staining of skin biopsies (5, 32) . Noticeably, the reduction of profilaggrin is more prominent at the protein than the mRNA level, with keratinocytes of IV-affected individuals showing a 30-60% reduction in steady-state mRNA and more than 90% reduction in protein ( Fig. 2and Fig. 3). This may reflect multiple subtle controls of profilaggrin gene expression during terminal differentiation.
Nuclear run-on assays have been used to demonstrate elongation of preinitiated RNA in various systems. Profilaggrin gene transcription as measured by run-on analysis was normal in keratinocytes from IV-affected individuals, while accumulation of the mRNA was reduced. There is also no evidence to suggest that transcriptional pausing or premature termination is relevant in the control of the profilaggrin gene in IV. The results suggest that the steady-state level of profilaggrin mRNA is not solely regulated at the level of mRNA synthesis, but additional posttranscription controls exist as well. This regulation appears to be specific to profilaggrin mRNA because the other mRNAs studied (K1, loricrin, glyceraldehyde-3-phosphate dehydrogenase, and HS26 ribosomal protein) were similar in keratinocytes from IV-affected individuals and normal controls.
Our study eliminates the possibility that the defective profilaggrin expression observed in IV occurs at the level of transcription. The results, however, do not preclude the possibility that IV results from mutation(s) in the profilaggrin gene. Mutations at the cap site, splice junction, initiation, or termination codon and coding region that result in low mRNA level and phenotypic aberrations have been documented in a number of human genetic diseases(33) . Results from sequence analysis of the 5`-, and 3`-noncoding, and flanking sequences suggest that these regions of the profilaggrin gene from IV-affected individuals are similar to normal. However, we have not sequenced the intron/exon boundaries, the extensive first intron, the filaggrin repeats or the sequence representing the unique leader or tail peptides. Nonsense or frameshift mutations in a variety of human genes are associated with significant reduction in the steady-state level of mRNAs. In numerous cases, the cytoplasmic rather than nuclear mRNAs appear to be unstable(34) . The mutation could also be allele-specific. It is possible that IV-affected individuals have one mutant profilaggrin allele in which a deletion or point mutation resides. However since IV is an autosomal dominant disease, if this were the case it would also mean that in the heterozygous condition the mutant allele would somehow inhibit the expression of its normal counterpart. This situation has been suggested in human diabetes insipidus (35) and type I angioneurotic edema(36) , which are also autosomal dominant diseases.
Posttranscriptional control regulates a number of
eukaryotic mRNAs and may take place at nuclear RNA processing/turnover,
nucleocytoplasmic transport, cytoplasmic mRNA turnover, or at
translational efficiency (for reviews, see (37) and (38) ). Analysis of available noncoding sequences of vertebrate
genes demonstrates the presence of many highly conserved sequences in
5` and 3` noncoding regions, suggesting a possible involvement of such
sequences in posttranscriptional control (39) . To date, most
studies suggest the 3`-noncoding region contains the primary
determinants of mRNA instability. Rapid turnover of histone RNA is
mediated by a sequence capable of forming a short stem-loop structure
at the extreme 3`-end of the nonpolyadenylated histone mRNAs (40) . In the cases of lymphokine (i.e. granulocyte
macrophage colony stimulating factor), cytokine (i.e. human
interferon-), and proto-oncogene (i.e. c-myc), a
variable number of AU-rich domains of the 3`-noncoding region confer
mRNA instability (for review, see (37) and (41) ).
This domain may interact with the poly(A)
tail(31, 42) , a 32-kDa polypeptide(43) , or a
complex of proteins (37, 43) prior to undergoing rapid
mRNA degradation. For granulocyte macrophage colony stimulating factor
mRNA, the 3`-noncoding region contains two regions that are responsible
for mRNA stabilization mediated by calcium(44) . The main
region maps to the AU-rich sequences. A second region located upstream
of the AU-rich domain contributes to the overall calcium response.
Active ongoing translation also plays an important role in
posttranscriptional control (45, 46) . Regulation of
mRNA stability may also be mediated by specific sequences other than
the AU pentamer. The paradigm is the presence of specific recognition
sites that may play dual functions for endonucleolytic cleavage and
protection of mRNA in Xenopus and Drosophila(47) . In addition, a repeated
pyrimidine-rich motif present in the 3`-noncoding region of
15-lipoxygenase mRNA may play critical role in translational regulation
during red blood cell differentiation(48) . These studies
clearly indicate a complex array of interaction between cis- and trans-acting factors in posttranscriptional
regulation.
The 3`-noncoding region of the human profilaggrin gene contains a single AUUUUUA and two AUUUUA sequences, similar to the consensus destabilizing AU pentamer (AUUUA). These sequences are present in the genes from both normal and IV-affected cells. Therefore, while this sequence may regulate profilaggrin mRNA degradation, the sequence alone is unlikely to be the cause of lower profilaggrin mRNA levels in IV. Recent studies identified at least three distinct cellular AU-binding factors that may mediate the pathway of RNA stability in other systems (49, 50) . Perhaps, a similar factor(s) functions as a trans-destabilizing element that specifically targets profilaggrin mRNA for increased degradation in keratinocytes from IV-affected individuals. Alternatively, a cellular factor(s) that selectively recognizes and stabilizes the profilaggrin mRNA in trans may be defective in keratinocytes from IV-affected individuals.
The reduced profilaggrin mRNA level in
keratinocytes from IV-affected individuals may also be a result of
defective regulation through protein kinase C. Keratinocytes express a
variety of PKC isoforms, and PKC induces profilaggrin and loricrin
mRNAs in mouse keratinocytes (51) . Additionally, changes in
the expression of different PKC isoforms are observed during
keratinocyte differentiation (52) and in psoriasis (52, 53) . nPKC- is the predominant isoform
expressed in epidermis and has been localized to the granular layer of
human epidermis(54) . The isoform may be involved in a
regulatory pathway controlling profilaggrin mRNA stability. In a study
on tumor necrosis factor mRNA decay, inhibition of PKC affected an
early step in the process of mRNA degradation by increasing the rate of
poly(A) removal(55) . Similar events may occur in IV; failure
of an element in the PKC pathway may lead to increased RNA instability.
The fact that profilaggrin probably functions as a calcium binding
protein in vivo suggests a close relationship with the known
calcium dependence of profilaggrin expression as well as epidermal
differentiation in
vitro(4, 56, 57) . Perhaps the putative
calcium binding domain of profilaggrin is involved directly with the
regulation of steady-state profilaggrin mRNA or translation. The
calcium binding domain of profilaggrin may also mediate autoregulation
at the posttranscriptional level similar to -tubulin (58) or at the transcriptional level as seen in the collagen
type I gene(59) .
The decrease or absence of profilaggrin in keratinocytes from IV-affected individuals would not only result in the absence of profilaggrin as a keratin aggregating protein but also as a calcium binding domain. The abnormal keratohyalin granules in IV suggests that intracellular calcium may not be sequestered in keratohyalin granules as is likely in normal cells. This could provoke defective calcium-dependent regulation of terminal differentiation resulting in the clinical phenotype. Alternatively, the clinical findings in IV may relate to the proposed water binding properties of free amino acids catabolized from filaggrin in the upper stratum corneum(60) . Decreased water binding capacity may result in abnormal desquamation and clinical scaling.
Our current results provide new molecular evidence that selectively impaired posttranscriptional control intrinsic to keratinocytes from IV-affected individuals is largely responsible for the reduction of the profilaggrin mRNA and protein in the disorder. The finding of such a molecular defect contributes to our understanding of the disorder as well as gene regulation during normal epidermal differentiation. Nonetheless, whether the low level of profilaggrin expression is a cause or a result of ichthyosis vulgaris remains in question and serves as the basis for further investigation.
Autoradiographic analysis in this study was carried out by the Phosphorimager Facility of the Markey Molecular Medicine Center at the University of Washington.
The biopsies were conducted at the Clinical Research Center facility of the University of Washington supported by the National Institutes of Health National Center for Research Resources Grant M01RR00037.