[Frontiers in Bioscience 1, a16-24, January 1, 1996]


Dean 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.

Table 1. The Effects of Calcium on Beta-galactosidase and Luciferase Reporter Activities.
Vector DNA0.03 mM Calcium1.2 mM Calcium
pGL3-basic247 ± 59358 ± 55
pGL3-control151,000 ± 24,000145,000 ± 16,000
pRSVbgal123,000 ± 14,000166,000 ± 61,000

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.

Figure 1: Effects of calcium on involucrin mRNA in NHK NHK were initially cultured in 0.03 mM calcium. The concentration of calcium was switched to the indicated calcium concentration for 24 hours before the cells were harvested. Twenty µg total RNA was fractionated in a 1% agarose gel, transferred onto nylon membrane and probed with 32P random primed-labeled involucrin cDNA probe. The blot was quantitated by densitometry. Data were normalized to the amount of the involucrin mRNA in the cells maintained in 0.03 mM calcium. Acridine Orange staining of the 28S and the 18S RNA is shown above the northern blot for the involucrin mRNA.

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.

Figure 2: Nuclear run-on assay of NHK. NHK were cultured in Clonetics KGM supplemented with 0.03 or 1.2 mM calcium in the presence of 32P-UTP. The newly synthesized RNA was then hybridized to pGEM-3Z containing the involucrin cDNA, GAPDH cDNA, and cDNA of 18S RNA as described in methods. The vector pGEM-3Z was used as the negative control.

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.

Figure 3: The 3.7 Kbp human involucrin promoter in pGL3-basic vector. The involucrin promoter was cloned in front of the luciferase reporter gene in pGL3-basic vector. The 3.7 Kbp of the involucrin gene contains 2476 bp of the 5' untranscribed region, the first exon, and the first intron. The locations of the CRE site, the proximal and distal AP-1 sites, the SP-1 site, the TATA box, and the transcription start site are shown.

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.

Figure 4: Deletion analysis of the involucrin promoter. The content of the deletion constructs is shown on the left, and their activity is shown on the right. Luciferase and beta-galactosidase were measured in lysates of keratinocytes transfected with the involucrin promoter sub-cloned into the pGL3-basic vector. The average ratios of their activities standard error of the mean (SEM) from at least 3 determinations were normalized with the average activity of construct A in cells cultured in 0.03 mM calcium. Where the error was less than the thickness of the line, no error bar is indicated. The open bars indicate activity ratios for cells maintained at 0.03 mM calcium, whereas the filled bars indicate the activity ratios for cells switched to 1.2 mM calcium for 24 h before they were harvested. The pGL3-basic vector without any insert was used as negative control (Vector). Construct A contains the sequence of the involucrin promoter between -2476 and +1228, and constructs B and C are 3' deletions of this construct at -182 and +3, respectively. A-1880, A-976, A-797, and A-156 were made from construct A, by removal of the 5' end of the base number indicated at unique restriction enzyme sites. Similarly, removal of the sequence between -2476 and -976 of the construct C resulted in construct C-976. Constructs D and E have internal deletions from -1000 to -156 and from -1880 to -156, respectively.

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.

Figure 5: Deletions analysis of 5' flanking region of the involucrin promoter. Luciferase and beta-galactosidase activities were measured as described in the legend to figure 4. The cells were transfected with construct F and 5' truncation mutations F-2406, F-2198, F-2177, F-2131, F-2028, and F-1947. Note the difference in scale between figures 4 and 5.

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).

Figure 6: Partial DNA sequence of the human involucrin promoter. AP-1 and SP-1 sites are enclosed by boxes. The direct repeat motif of GGCAGA is underlined. Regions of human keratin-1 that are homologous to the human involucrin promoter are in bold, and the locations of these sites are indicated. Locations of the 5' end of the construct F-2431 and F-2028 are also indicated.

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