©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Decreased Profilaggrin Expression in Ichthyosis Vulgaris Is a Result of Selectively Impaired Posttranscriptional Control (*)

(Received for publication, May 31, 1994; and in revised form, November 2, 1994)

Wilas Nirunsuksiri (1) Richard B. Presland (1) (3) Steven G. Brumbaugh (1) Beverly A. Dale (1) (3) (2) Philip Fleckman (1)(§)

From the  (1)Division of Dermatology, Department of Medicine, and Departments of (2)Biochemistry and (3)Oral Biology and Periodontics, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Ichthyosis vulgaris (IV) (^1)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.


MATERIALS AND METHODS

Diagnosis of Ichthyosis Vulgaris

Probands were identified from patients seen in the University of Washington Medical Center Dermatology Clinics and from individuals referred by clinical dermatologists from the community. Subjects who met published clinical criteria for IV (1, 11) were biopsied from the extensor surface of the arm after obtaining informed consent. Biopsies were fixed in methyl Carnoy's for light microscopy and immunocytochemistry and fixed in half-strength Karnovsky's for electron microscopy as described previously(12) . Individuals with the clinical criteria for IV who had one or fewer layers of granular cells in hematoxylin and eosin-stained sections(1) , who had absent or attenuated staining with the anti-profilaggrin antibody AKH1 with normal staining with the anti-keratohyalin antibody AKH2 (13) and who had no keratohyalin granules when examined by electron microscopy (3, 5) were considered to be affected. In this report, results from affected, unrelated individuals from three different families are presented. Related, unaffected family members served as controls for two cases, and an unrelated, age- and sex-matched subjects for the other.

In Situ Hybridization

Biopsies were obtained from the extensor surface of the arm and snap frozen in Tissue-Tek OCT (Miles, Inc.) embedding medium. Ten-micron frozen sections were fixed briefly in paraformaldehyde and processed by standard techniques (14) with the following modifications; proteinase K digestion was omitted, an initial 1-h wash at room temperature with 4 times SSC (1 times SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0) was added after hybridization, and 10 mM dithiothreitol was added to all rinses. Hybridization was with a S-labeled riboprobe generated from a human filaggrin repeat cloned into pGEM-1 (Promega Corp.). The plasmid was linearized with EcoRI, and antisense riboprobe was generated with the use of T7 RNA polymerase in the presence of [alpha-S]UTP (1,000 Ci/mmol) (DuPont NEN).

Cell Culture

Adult human keratinocytes were obtained from blister biopsies as described previously(6) . Keratinocytes were cultured on mitomycin C-treated 3T3 cells and maintained in a humidified 5% CO(2) atmosphere at 36.9 °C in Dulbecco's modified Eagle's medium (Life Technologies) containing 20% fetal calf serum, hydrocortisone, cholera toxin, and epidermal growth factor. Cells were fed 3 times a week and 24 h before harvesting. To ensure profilaggrin expression, cells were harvested 3 days after cells reached confluence.

Protein Extraction, SDS-PAGE, and Western Blotting

Total urea-Tris-soluble protein extracts (15) were obtained from keratinocytes cultured in parallel with those used for the isolation of RNA and nuclei. Equal protein loadings were separated on discontinuous 7.5-15% SDS-PAGE gels, and proteins were blotted to nitrocellulose (Schleicher and Schuell). Profilaggrin was detected with polyclonal anti-human profilaggrin/filaggrin antiserum(16) , K1 with monoclonal antibody AE3 (a generous gift of T.-T. Sun, New York University Medical Center, (17) ), and loricrin with antibody raised against a synthetic peptide corresponding to the 14 most carboxyl-terminal residues of mouse loricrin known to cross-react with human loricrin(18) .

Plasmids, Probes, PCR Primers, and Genomic DNA

1) The profilaggrin coding probe was a 972-bp filaggrin repeat of exon 3(7) .

2) The profilaggrin 5` upstream region (885 bp) was generated from human genomic DNA using the following oligonucleotides^2 ((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) .

Northern Analysis

Total cellular RNA was prepared from cultured keratinocytes using the guanidine thiocyanate-acid phenol method(22) . Equal amounts of total RNA (10 µg) were separated on 1% glyoxal gels, blotted onto GeneScreen Plus membranes (DuPont NEN) and hybridized to a nick-translated or random primed probe overnight at 60 °C in the buffer recommended by the manufacturer except that 200 µg/ml sonicated denatured salmon sperm DNA was included. Filters were washed 3 times in 2 times SSC, 0.1% SDS, followed by 3 times in 0.1% SDS/0.1 times SSC at 65 °C. All Northern blots were reprobed with glyceraldehyde-3-phosphate dehydrogenase and HS26 cDNA to ascertain equal RNA loadings. The autoradiographs were scanned using the PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and quantified using image analysis software (ImageQuant Version 3.22, Molecular Dynamics). All comparison between hybridization signals was based on equal glyceraldehyde-3-phosphate dehydrogenase or HS26 signal.

Isolation of Nuclei and Nuclear Run-on Assays

First or second passage human keratinocytes were cultured to 3 days post-confluence. Nuclei were isolated by a modification of the method of Greenberg and Bender(23) . Keratinocytes were harvested from five to six 100-mm plates, resuspended in 1% Nonidet P-40 lysis buffer, vortexed briefly, and severed with sterile scissors. Cells were broken using 40 strokes of a Dounce homogenizer, and nuclei were isolated, snap frozen in liquid nitrogen, and stored at -70 °C. Nuclei were incubated with run-on reaction buffer in the presence of 250 µCi of [alpha-P]GTP (3,000 Ci/mmol) for 30 min. Labeled RNA was isolated and purified through a SuperSelect column (5` 3`, Boulder, CO). Equivalent P-labeled RNA (as determined by trichloroacetic acid precipitation) was hybridized to plasmid DNA that had been linearized, denatured, and immobilized onto nitrocellulose using a slot blotting apparatus (Bio-Rad). Hybridization was carried out in 1 ml of TES buffer with 100 mg/ml denatured sonicated salmon sperm DNA for 48-72 h at 65 °C, followed by two washes in 2 times SSC, 0.1% SDS and two washes in 0.1 times SSC, 0.1% SDS at 65 °C for 30 min. Exposure to x-ray film was for 3-7 days. Nuclear run-on was also carried out in the presence of 0.5% sarkosyl (24) in order to distinguish between polymerase initiation and elongation. alpha-Amanitin (2 µg/ml) was used to show that in vitro transcription was due to RNA polymerase II activity(24) .


RESULTS

In Situ Analysis of Profilaggrin mRNA in IV and Normal Epidermis

It has been shown previously by immunohistochemical techniques using an anti-human profilaggrin antibody that immunoreactive profilaggrin and filaggrin are markedly decreased in the epidermis of IV-affected individuals compared with normal controls(5) . To determine if the decrease is due to a reduction in profilaggrin mRNA, in situ hybridization using a S-labeled riboprobe transcribed from a human filaggrin repeat was performed. A dramatic decrease in silver grains in the granular layer of affected IV epidermis compared with normal skin was seen (Fig. 1) showing reduced levels of profilaggrin mRNA in the epidermis of IV-affected individuals and confirming studies at the protein level (5) . No significant labeling of the epidermis was seen with the sense strand riboprobe (data not shown).


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.



Decreased Profilaggrin Expression in IV Keratinocytes

Western analysis of profilaggrin, K1, and loricrin in protein extracts from keratinocytes obtained from IV-affected individuals compared with normal controls is demonstrated in Fig. 2. It should be noted that cultured human keratinocytes express profilaggrin at confluence but do not process it to filaggrin as normally occurs during terminal differentiation in vivo(6) . It typically appears as a broad smear on SDS gels. The anti-human profilaggrin antibody detected very little profilaggrin in extracts from affected individuals, in agreement with previous findings(6) . We visually estimated the amount of immunoreactive protein in keratinocyte extracts from affected individuals to be less than 10% of normal controls. This result paralleled the loss or reduction of keratohyalin granules observed in the corresponding skin biopsies (data not shown). In contrast, the expression of neither loricrin, another marker of the late stages of epidermal differentiation also localized in keratohyalin granules, nor K1, a marker of suprabasal differentiation, were reduced (Fig. 2). The results strongly suggest that the defect in keratinocytes obtained from IV-affected individuals is specific to profilaggrin.

Steady State Level of the Profilaggrin mRNA in IV Keratinocytes

To gain insight into the etiology of the disease, profilaggrin mRNA levels in keratinocytes from affected individuals and controls was determined. Northern analysis of total RNA harvested from confluent keratinocytes of three unrelated IV subjects and controls was conducted with a probe from a filaggrin repeat within exon 3 of the profilaggrin gene (Fig. 3A). The level of profilaggrin mRNA in affected cells was markedly reduced compared with the normal counterparts (Fig. 3B). Similar results were obtained when exon 2, which contains part of the EF-hand domain, was used as a probe (data not shown). The intensity of each hybridization signal was quantified using the PhosphorImager and the level of mRNA normalized to the internal glyceraldehyde-3-phosphate dehydrogenase control. The levels of profilaggrin mRNA were 30, 61, and 43% of their corresponding controls (from left to rightpanel). When loricrin- and K1-specific probes were used on the same blots, no differences in RNA levels were seen between normal and affected (Fig. 3B). These results were consistent with those of the Western analysis, although compared with the normal counterparts, the levels of immunoreactive profilaggrin protein were much lower in IV keratinocytes than the levels of profilaggrin mRNAs.

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.

Transcription of Profilaggrin mRNA in IV and Normal Keratinocytes

Comparison of profilaggrin mRNA levels using Northern blots determines the steady state level of RNA but does not differentiate between decreased RNA levels resulting from defective transcriptional or posttranscriptional events (see ``Discussion''). To delineate between the two, profilaggrin transcription was examined by nuclear run-on assay.

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. (^3)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.



Analysis of 5`-Flanking Region from IV-affected Individuals

The sequence of the 5`-upstream flanking DNA of human profilaggrin from -1 to -869 bp obtained from three IV-affected individuals was also determined to identify any promoter mutations that might account for the lower profilaggrin mRNA expression. Very few differences in nucleotide sequence were apparent (data not shown); no changes were specific to IV. Such identified changes could be due to allelic polymorphism or errors due to PCR. No changes were seen in the immediate upstream region containing putative regulatory motifs such as the TATA box, SP1, or AP1 sites(7, 8) .

Analysis of 3`-Noncoding Region

A number of genes whose expression is reduced under various conditions contain AU-rich sequences that act as destabilizing signals in the 3`-noncoding region of the mRNA(30, 31) . AU-rich sequences are found in the 3`-noncoding region of the human profilaggrin gene(9) . Therefore, the 3`-noncoding and flanking sequences of the profilaggrin gene in IV-affected individuals were investigated for possible alterations that could cause decreased profilaggrin mRNA stability.

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.


DISCUSSION

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-beta), 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 beta-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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants PO1 AM 21557 and R37 DE 04660, the Endowed Dermatology Research Fund, and the Hammock Trust. 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: Div. of Dermatology, RM-14, University of Washington, Seattle, WA 98195. Tel.: 206-543-5290; Fax: 206-543-2489.

(^1)
The abbreviations used are: IV, ichthyosis vulgaris; bp, base pair(s); PCR, polymerase chain reaction; HS26, human S26 ribosomal protein; PAGE, polyacrylamide gel electrophoresis; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.

(^2)
R. B. Presland, unpublished data.

(^3)
W. Nirunsuksiri and P. Fleckman, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to Paul V. Haydock for his early contribution to the work and the riboprobes. We thank Barbara Hager for her excellent technical assistance in tissue culture and PAGE, and Mary Hoff for her expertise in in situ hybridization. T.-T. Sun and Dennis Roop generously provided AE3 and loricrin antibodies, respectively. We also thank Phillipe Fort for the cDNA for HS26.

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.


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