|[Frontiers in Bioscience 1, a16-24, January 1, 1996]|
REGULATION OF INVOLUCRIN GENE EXPRESSION BY CALCIUM IN NORMAL HUMAN KERATINOCYTESDean C. Ng, Mei-Jhy Su, Richard Kim, and Daniel D. Bikle.
Endocrine Unit, VA Medical Center, University of California, 4150 Clement Street, 111N, San Francisco, CA 94121.
Received 12/7/95; Accepted 12/29/95; On-line 1/1/96
Calcium treatment (1.2 mM) did not substantially affect the luciferase activity of the vectors pGL3-basic, pGL3-control, or pRSVbgal. Pre-confluent NHK were transfected with each vector and were either maintained in 0.03 mM calcium or switched to 1.2 mM calcium for 24 hours. The luminometer readings in relative light units (RLU) of cells were listed in Table 1. The variability was most likely due to difference in transfection efficiency, which range from 8 to 15%. Mock transfected cells showed light output less than 2-fold above the assay blank. Cells transfected with the pGL3-basic vector showed low luciferase activities compare to the pGL3-control vector, but were 10 to 15-fold higher than the mock transfected cell extract.
Involucrin mRNA levels were low in the pre-confluent cells maintained at 0.03 mM calcium. Pre-confluent NHK cultured in 0.03 mM CaCl2 were supplemented with CaCl2 to a final concentration of 0.07, 0.1, 0.3, and 1.2 mM for 24 hrs. Cells maintained in 0.03 mM CaCl2 This experiment was performed a total of three times and a representative gel and northern blot are shown in figure 1. In all cases, calcium increased the involucrin mRNA levels in a dose-dependent manner with the maximum effect at 1.2 mM CaCl2.
To determine if the effect of CaCl2 on the involucrin mRNA level was due to increased transcription, nuclear run-on assays were performed on cells cultured at 0.03 mM CaCl2 and cells incubated with 1.2 mM CaCl2 for 24 hrs. Newly synthesized mRNA was labeled with 32P-UTP and hybridized to immobilized pGEM-3Z plasmids containing the involucrin cDNA, GAPDH cDNA, 18S RNA cDNA, and vector alone without insert. Non-specific targets hybridized to the membrane were removed by successive washes, and the resulting blot was exposed to film. This experiments were carried out three times and a representative experiment is shown in figure 2. Densitometry of the autoradiogram indicates that the transcription rate of the cells treated with 1.2 mM CaCl2 is several-fold higher than the rate of synthesis in the untreated cells (n=3). The calcium induced increase in involucrin mRNA synthesis was specific; neither GAPDH nor 18S RNA synthesis was affected by calcium. The pGEM-3Z vector, as expected, did not bind the newly formed transcripts.
To determine if the increase in mRNA transcription was due to a genomic element in the involucrin promoter, a 3.7 Kbp fragment of the involucrin gene (3,4) was sub-cloned into the pGL3-basic luciferase reporter vector. This 3.7 Kbp fragment contains 2476 bp of 5' upstream region, the first exon of 43 bp that contains a non-coding region of the involucrin mRNA, and the first intron of 1188 bp (Figure 3). All the coding sequence of the involucrin gene is located in the second exon, and is not part of the 3.7 Kbp construct. Sequence analysis revealed one CRE site at -2441 and five putative AP-1 sites. Two of the AP-1 sites have been shown to be regulated by phorbol esters (18) and are noted on figure 3.
NHK transfected with the 3.7 Kbp involucrin construct (Figure 4, construct A) exhibited an 8-fold induction of the luciferase/beta-galactosidase activity ratio when incubated with medium containing 1.2 mM CaCl2 for 24 hours, compared to cells that were maintained in medium with 0.03 mM CaCl2.
Deletion of the first intron of the human involucrin gene at +186 (B) and -3 (C) did not affect the calcium-dependence but did enhance the basal luciferase reporter activity (Figure 4). Thus, intron A contains a suppressor element of basal activity but no calcium-responsive element. A series of 5' deletion mutations using unique restriction sites within the involucrin gene at -1880, -976, -797, and -156 was then constructed and tested (Figure 4). In all these constructs, minimal reporter activities were detected, regardless of calcium concentration indicating that the region between -2476 and -1880 of the human involucrin promoter is required for basal and calcium-induced transcriptional activity.
Results with these constructs prompted the construction of two internal deletion mutations with deletions from -1880 to -156 (D) and from -1000 to -156 (E). Since the reporter activity of the construct with the deletion between -1880 to -156 (D) was responsive to calcium, a calcium-responsive element must be located between -2476 and -1880 (Figure 4). Furthermore, deletion of -1880 to -156 enhanced the basal activity by 34-fold. On the other hand, no enhancement was observed when only -1000 to -156 was removed, indicating the presence of a suppressor element between -1880 and -1000.
Since the region between -1000 and -156 and the first intron did not show calcium dependence, these regions were removed from the original involucrin promoter construct (A) to yield construct F (Figure 4). The calcium responsive region between -2476 and -1880 was then examined by a series of 5' deletions. The results of deletions at -2406, -2198, -2177, -2131, -2028, and -1947 are shown in figure 5. Between -2476 and -2131, each of the four deletions caused successive reductions in basal transcription activity, while the transcription of each could still be stimulated by calcium. Therefore, multiple calcium-independent enhancer elements exist in this region. While deletion of -2131 to -2028 had no apparent effect on the basal activity, it reduced the calcium-induced activity greatly. Thus a calcium-dependent element is present in this region of 103 bp.
The sequence of this 103 bp region is shown in figure 6. The sequence revealed an AP-1 site (-2116 to -2110), a SP-1 site (-2107 to -2102), a novel direct repeat (-2099 to -2085), and two regions that share homology with a 422 bp region of the human keratin-1 3' flanking region that has been previously demonstrated to contain calcium-dependent elements (19). The sequence of the first region (CTTGATCTG) is identical to the base number 20 to 38 of the human keratin-1 3' region. The second region (GTGCCCCAGA) has a 90% homology to a different region of the human keratin-1 gene (position 181 to 190).