Molecular cloning of a novel thyroid hormone-responsive gene, ZAKI-4, in human skin fibroblasts.

Utilizing a method called "differential display of mRNAs by means of polymerase chain reaction", the cDNA fragment of a thyroid hormone-responsive gene ZAKI-4 was cloned from cultured human skin fibroblasts. Northern blot analysis revealed that there were two ZAKI-4 mRNA species (3.4 and 1.4 kilobases (kb)), and they were up-regulated by a physiological concentration of triiodothyronine (T3). This T3 effect was abolished by the treatment with cycloheximide, indicating the possibility that gene ZAKI-4 is regulated by T3 in an indirect fashion, through an intermediate product of T3, rather directly by T3 itself. No effect of T3 on ZAKI-4 mRNA stability suggested that T3 induces the mRNA at the transcriptional level. Rapid amplification of cDNA ends confirmed the presence of two mRNA species. ZAKI-4 mRNA was detected in heart, brain, liver, and skeletal muscle but not in placenta, lung, kidney and pancreas. In skin fibroblasts and skeletal muscle, 3.4-kb mRNA was the major species, whereas 1.4-kb mRNA was dominant in heart, brain, and liver. The sequence analysis suggested that the two mRNA species arise from alternative polyadenylation and code a single protein of 192 amino acids. No homologous protein sequence was found in a data base. Elucidation of the function of ZAKI-4 gene product will provide new insights into an important role of T3 in various organs.

Thyroid hormones (thyroxine and triiodothyronine (T 3 )) 1 play a vital role in fetal development and throughout life in humans. T 3 , an active form of the thyroid hormones, exerts its effect through binding to its nuclear receptor and regulating expression of target genes. Thus, identification of T 3 -responsive genes in various tissues is important to elucidate T 3 action at molecular and cellular levels in humans. However, the search for T 3 -responsive genes in human tissues is hampered by the difficulty of obtaining tissues from subjects in various thyroid states. Clonal cell lines established from malignant tissues could be used to identify T 3 -responsive genes. However, they might aberrantly respond to hormones (1,2). Identification of T 3 -responsive gene(s) from the tissues that maintain differentiated function is thus preferable. Human skin fibroblasts fulfill this requirement since they express T 3 -receptors (3)(4)(5)(6) and are responsive to T 3 . In cultured skin fibroblasts, we have shown that T 3 inhibits the synthesis of glycosaminoglycan (7,8) and fibronectin (9), and Chait et al. (10) demonstrated that it enhances low density lipoprotein degradation. These effects of T 3 were used for the tissue diagnosis of generalized resistance to thyroid hormone (11).
Despite the fact that cultured human skin fibroblasts are responsive to T 3 , few T 3 -responsive genes have been cloned. Recently, Liang and Pardee (12,13) developed a method called "differential display of mRNA by means of the polymerase chain reaction (PCR)" to identify and analyze altered gene expression at the mRNA level in any eukaryotic cells. A similar method was developed by Welsh et al. (14,15). We have chosen the method to clone T 3 -responsive genes in cultured skin fibroblasts, since it has been successfully employed to identify transcripts specific for human tumors (13).
We identified a T 3 -responsive gene expressed in cultured human skin fibroblasts. The expression of the gene termed ZAKI-4 is positively regulated by a physiological concentration of T 3 . Complete cDNA sequence obtained by rapid amplification of cDNA ends (16) revealed that the gene is transcribed into two mRNA species, both of which code a single peptide of 192 amino acids.

MATERIALS AND METHODS
Cell Culture and Treatment-Skin fibroblasts were obtained by punch biopsies from the deltoid region of four healthy subjects (two men and two women, ranging in age from 9 to 37 years old (males 9 and 29 years, females 25 and 37 years)). The fibroblasts were used between the fourth and the ninth passages after the initial plating and were grown to confluence in 60-mm-diameter plastic culture dishes (Becton Dickinson Labware, Lincoln Park, NJ) with 4 ml of Dulbecco's modified Eagle's medium (DMEM, Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) containing 10% fetal bovine serum (Bioserum, Victoria, Australia) and 50 units/ml penicillin G (Life Technologies, Inc., Grand Island, NY) and 50 mg/ml streptomycin (Life Technologies, Inc.) at 37°C in an atmosphere of air, 5% CO 2 and 100% relative humidity. To study the effect of T 3 on ZAKI-4 mRNA, the cells were exposed to 4 ml of the same medium in which fetal bovine serum was replaced with 10% bovine serum from a thyroidectomized calf (TxBS; Rockland Farms, Gilbertsville, PA) (7). After three days of culture, the medium was replaced with DMEM containing either 10% TxBS alone or TxBS plus T 3 (10 Ϫ10 -10 Ϫ8 M; Sigma). The cells were further incubated for the indicated periods of time. Total RNA was extracted from cells by the method of Chomczynski and Sacchi (17).
To examine whether the T 3 -mediated increase in ZAKI-4 mRNA * This work was supported in part by Grant-in-Aid for Scientific Research 07671125 from the Ministry of Education, Science and Culture of Japan (to Y. M.) and a research grant from Daiko Foundation (to H. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) D83407 (for ZAKI-4 cDNA sequence).
requires de novo protein synthesis, the medium of confluent fibroblast cultures was replaced with DMEM supplemented with 10% TxBS and 25 M cycloheximide (Wako Pure Chemical Industries, Osaka, Japan) was added 15 min before T 3 (10 Ϫ7 M) addition. The cells were further incubated for 24 h, and total RNA was extracted. The effect of T 3 on the stability of ZAKI-4 mRNA was evaluated as follows. Fibroblast cultures in confluency were treated with 10 Ϫ7 M T 3 for 72 h. Then the cells were washed two times with 4 ml of serumless DMEM. They were incubated in DMEM supplemented with 10% TxBS containing actinomycin D (SERVA, Heidelberg, Germany) at a final concentration of 5 g/ml with or without 10 Ϫ7 M T 3 . After further incubation for 12, 24, and 48 h, the cells were harvested for RNA extraction.
Differential Display of mRNA by PCR-Total RNA was extracted from the two fibroblast cell lines, a) male 29 year and b) female 25 year, incubated with or without T 3 for 24 h. Differential display was carried out using RNAmap Kit (GenHunter Corp., Brookline, MA). Briefly, 0.2 g of the total RNA was reverse transcribed with T12MA (5Ј-TTTTTTTTTTTTMA-3Ј, where M represents degenerate bases) and subsequently amplified by PCR using five different arbitrary primers , and AP-20 (5Ј-GT-GATCGGAC-3Ј) according to the supplier's protocol with minor modification. In the PCR [ 33 P]dATP (specific activity 70 TBq/mmol; DuPont NEN) was used instead of [ 35 S]dATP as recommended by Trentmann et al. (18).
After an initial 2-min denaturation at 94°C, PCR was carried out using AmpliTaq DNA polymerase (Perkin-Elmer) for 40 cycles with denaturation at 94°C for 30 s, annealing at 40°C for 2 min, and extension at 72°C for 30 s. An aliquot of each PCR product (5 l) was analyzed on a 5% DNA sequence gel, which was subsequently exposed to X-AR film (Eastman Kodak Co.) for autoradiography. The cDNA fragments of interest were recovered from the gel and subsequently reamplified with the same primer set and PCR conditions except that no isotopes were added. The reamplified cDNA fragments were cloned into pGEM-T vector from Promega (Madison, WI).
Northern Blot Analysis-Total RNA samples isolated from the four fibroblast cell lines cultured in the presence or absence of T 3 were subjected to Northern blot analysis as described previously (19).
For the analysis of tissue distribution of ZAKI-4 mRNA expression, human multiple tissue Northern blot (Clontech, Palo Alto, CA), premade Northern blot of 2 g of poly(A) RNA from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas, was used.
Rapid Amplification of cDNA Ends (RACE)-RACE (13), a procedure to amplify nucleic acid sequences from an mRNA template between a defined internal site and unknown sequences at either the 3Ј or 5Ј end of mRNA, was performed by utilizing a kit (Life Technologies Inc.) according to the supplier's protocol. To increase the fidelity of PCR, Ex Taq DNA polymerase (TaKaRa Biomedicals, Shiga, Japan) was used in RACE, since Ex Taq possesses 3Ј-5Ј exonuclease activity (3Ј-editing activity), resulting in the reduction of misincorporation per cycle as reported by Barnes (20).
Sequencing Analysis-DNA sequences from both strands were determined on an ABI 373A DNA sequencer at least four times. Sequence searches of GenBank TM was carried out using BLAST program (21).
In Vitro Translation-The complete cDNAs coding ZAKI-4 mRNAs (1.4 and 3.4 kb) were synthesized by reverse transcription-PCR and inserted into pGEM-T vector. In vitro transcription and translation were performed by using TNT coupled reticulocyte lysate systems (Promega) with EXPRE 35 S 35 S protein labeling mix (DuPont NEN), according to the manufacturer's instruction. The synthesized proteins were analyzed by 17.5% SDS-polyacrylamide gel electrophoresis, followed by autoradiography. Display  of mRNAs-Fig. 1A illustrates the differential display of mRNAs in human skin fibroblasts cultured in the presence or absence of T 3 . At a glance, the patterns of bands were similar between T 3 -treated and nontreated fibroblasts. The patterns between the two fibroblasts from different individuals were similar when the same set of primers was used. However, a closer look identified some bands whose densities were either increased or decreased in the fibroblasts cultured with or without T 3 (Fig. 1B). When the changes in the densities by T 3 were reproduced in the two fibroblasts from different individuals, the bands were selected as candidates of T 3 -responsive genes. Nine cDNA fragments were extracted from the gel, reamplified by PCR with the same set of primers, and subcloned into pGEM-T vector. The cloned cDNA fragments were used as the probes for Northern blot analysis to confirm that the expression of the putative T 3 -responsive genes was indeed regulated by T 3 . It was demonstrated that the expression of only one gene (ZAKI-4) was up-regulated by T 3 . The mRNA for the rest of the genes were either undetectable or unaltered by the addition of T 3 (data not shown). As shown in Fig. 2, a significant increase was observed 24 h after the addition of 10 Ϫ10 M T 3 , a physiological concentration in the presence of 10% TxBS (7). The mRNA was further increased by the addition of 10 Ϫ8 M T 3 . Also shown in the figures was the existence of two species of ZAKI-4 mRNA. In addition to a dominant band of 3.4 kb, a faint band of 1.4 kb was observed. The two mRNA species were increased proportionately by T 3 . As illustrated in Fig. 3, ZAKI-4 mRNA levels increased 12 h after the addition of T 3 (10 Ϫ8 M). These results were reproduced with four different human skin fibroblasts obtained from different individuals (data not shown).

Identification of T 3 -responsive Genes by Differential
Mechanism Involved in the Induction of ZAKI-4 mRNA by T 3 -As shown in Fig. 4, the increase in ZAKI-4 mRNA was completely blocked by the treatment with cycloheximide. By using actinomycin D the half-life of ZAKI-4 mRNA was studied (Fig. 5). It was demonstrated that T 3 did not affect the stability (t 22 h in the absence and 20 h in the presence of T 3 ). These results indicate that T 3 induces ZAKI-4 mRNA at the transcriptional level, but de novo protein synthesis is required for the induction.
Tissue Distribution of ZAKI-4 mRNA-The expression of ZAKI-4 mRNA was evident in the poly(A) RNAs from heart, brain, liver, and skeletal muscle (Fig. 6). However, no mRNA band was detected in the poly(A) RNAs from placenta, lung, kidney, and pancreas. Thus, ZAKI-4 mRNA is not only expressed in the fibroblasts but also in various T 3 -responsive organs.
Note that dominant ZAKI-4 mRNA in skeletal muscle as well as in fibroblasts is 3.4 kb in size, whereas it is 1.4 kb in the other organs.
Determination of Full-length Nucleotide Sequence of ZAKI-4 cDNA-The entire nucleotide sequence of ZAKI-4 cDNA is shown in Fig. 7. As illustrated at the top of the figure, the original cDNA isolated from the differential display was ap- proximately 180 base pairs long. To isolate the full-length cDNA, RACE was employed (primer design was indicated in the figure). 5Ј-RACE produced a product extending 680 base pairs from the 5Ј-end of the original clone. On the other hand, 3Ј-RACE produced two products, one extending approximately 200 base pairs from the 3Ј-end of the original clone and the other extending 2,300 base pairs. Two products produced by 3Ј-RACE suggested the presence of two mRNA species with different 3Ј-ends. Alignment of the sequences enabled us to delineate the entire cDNA sequence. Putative polyadenylation signals could be assigned at nucleotide positions 1023 AT-TAAA 1028 and 3168 AATATA 3173 , giving rise to two mRNA species. The search for an open reading frame revealed that both short and long species of ZAKI-4 mRNAs code a single polypeptide. In frame termination codon 160 TAA 162 was followed by two initiation codons. The presence of two ATG codons for methionine at N-terminal ends suggested that ZAKI-4 mRNAs may code a protein of either 192 or 197 amino acids. Since Kozak's sequence (22) is present 5Ј-upstream of the second ATG codon, translation initiation likely occurs at the second ATG, giving rise to a protein of 192 amino acids. Proline and valine residues were abundant in the molecule.
From the cDNA sequence, it was also deduced that the original clone isolated from the differential display was amplified from the sequence 5Ј-684 CGACAGTGAC 693 -3Ј to 5Ј-848 TCAA-AAAAAA 857 -3Ј with the corresponding primer AP-16 (5Ј-CGT-CAGTGAC-3Ј) and T12MA (3Ј-AMTTTTTTTTTTTT-5Ј). Thus, there is only one mismatch between 5Ј-end sequence of the arbitrary primer, while 3Ј-primer T12MA did not hybridize with the actual cDNA ends but recognized eight adenylate sequences present in the nucleotide from 848 to 857.
Homology search of the ZAKI-4 sequence revealed that there FIG. 7. Nucleotide sequence of ZAKI-4 cDNA. The strategy for 5Ј-and 3Ј-RACE is depicted at the top. For 5Ј-RACE, first cDNA strand was synthesized from total RNA from human skin fibroblasts treated with T 3 using primer AS1. After tailing with poly(dC), the cDNA was amplified with a nested primer AS2 and a poly(dG) adapter primer. For 3Ј-RACE, cDNAs were prepared by reverse transcriptase using the same RNA used in 5Ј-RACE as a template and oligo(dT) adapter primer. ZAKI-4-specific cDNAs were amplified with primer S1 and an adapter primer. Primer sequences are as follows; AS1, 5Ј-819 AGAGGGACG-GCTATTATCG 800 -3Ј; AS2, 5Ј-797 AAG-GAGCAGGCAGCTCAGTT 778 -3Ј; S1, 5Ј-704 AAGAGGACCCAAAGACTTCC 723 -3Ј. In the nucleotide sequence, initiation and stop codons are indicated with boldface letters. The boxed sequence is the fragment obtained by the differential display. In the box, italic sequences with underlines correspond to the annealing sites for the primers used in the differential display. Putative polyadenylation signals and corresponding adenylation sites were also indicated. are at least two related sequences. Both of them are short sequences reported as expressed sequence tag (EST). The 5Јend (277-614) of ZAKI-4 cDNA is identical to the EST (NCBI accession number T09144) from 73 days postnatal female human brain (24). Another EST (NCBI accession T23571) from 3-month human infant brain is identical to the 3Ј-end (2939 -3187). Since the present study revealed that ZAKI-4 gene is also expressed in human brain, these two ESTs are likely to be a part of ZAKI-4 cDNA.
In Vitro Translation of ZAKI-4 mRNA-Both short and long species of mRNAs for ZAKI-4 contained an open reading frame as described above. To confirm that both mRNAs code a single peptide, each cDNA was synthesized by reverse transcription-PCR using RNA obtained from the fibroblasts cultured in the presence of T 3 and cloned into pGEM-T vector. The corresponding mRNAs were transcribed by T7 RNA polymerase and translated in rabbit reticulocyte lysate. As shown in Fig. 8, both mRNAs programmed the synthesis of a peptide with a molecular mass of 26 kDa, which roughly corresponds to the molecular weight estimated from the amino acid sequence. DISCUSSION The cloning of a T 3 -responsive gene ZAKI-4 in human fibroblasts was described. It was demonstrated that the increase of ZAKI-4 mRNA by T 3 is regulated at the transcriptional level, since the stability of the mRNA was not affected by T 3 . However, the T 3 effect requires de novo protein synthesis, suggesting the possibility that regulation of ZAKI-4 gene expression by T 3 is indirect. To date, very few T 3 -responsive genes have been cloned in human skin fibroblasts. Expression of fibronectin mRNA was suppressed by T 3 (23). The decrease in glycosaminoglycan (7,8) synthesis by T 3 suggested that expression of some genes involved in the synthesis could be repressed by the hormone. To our knowledge, ZAKI-4 gene is the only gene that is up-regulated by T 3 in human skin fibroblasts.
Northern blot analysis revealed the presence of two mRNA transcripts (3.4 and 1.4 kb) for ZAKI-4 gene. Since both mRNA species were proportionately increased by T 3 , it was suggested that both mRNAs were transcribed from the same gene. The data from 3Ј-RACE also suggested the presence of two mRNA species with different 3Ј-ends. From the sequence of the entire cDNA it is suggested that the two mRNA species originate from alternative polyadenylation, since putative polyadenylation signals could be localized at two sites with a distance of 2 kb, accounting for the size difference.
No homologous protein sequence to ZAKI-4 was found in the SWISSPROT data base. It is thus impossible to speculate about the function of ZAKI-4 gene product based on the sequence data. However, ZAKI-4 mRNA is expressed not only in fibroblasts but also in brain, heart, liver, and skeletal muscle, suggesting that thyroid hormone exerts its effect by up-regulating its expression in these organs.
There was a size difference in the predominant ZAKI-4 mRNA species in different organs. In skin fibroblasts and skeletal muscles, the mRNA of 3.4 kb in size was the major species, whereas 1.4 kb mRNA was dominant in brain, heart, and liver. Specific distribution of ZAKI-4 mRNA species with differing 3Ј-ends raises a possibility that there is a mechanism for tissue-specific regulation of alternative polyadenylation.