|[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
Extracellular calcium increases involucrin mRNA levels in NHK in a dose-dependent manner. Results from nuclear run-on experiments indicate that calcium increases the transcription rate of the involucrin gene. Thus, the involucrin gene in NHK is transcriptionally regulated by extracellular calcium.
Deletion studies using unique restriction enzymes to remove large fragments of DNA from the involucrin promoter revealed the presence of repressor elements in the region between -1880 and -1000 and in the first intron. This approach also demonstrated the existence of a calcium-dependent element between -2476 and -1880. More detailed study of this region with additional deletions refined the location of the calcium-dependent element to a region of 103 bp from -2131 to -2028.
A putative AP-1 element (-2116 to -2110) is present within this region. The AP-1 element was first shown to be inducible by phorbol esters (9,10). This element has also been demonstrated to be regulated by cAMP and calcium in neural and endocrine cells (11,12). Therefore, calcium may induce expression of the involucrin gene through the AP-1 site. In addition, there is a SP-1 site (-2107 to -2102) and a novel direct repeat (GGCAGA NNN GGCAGA) that may also play a role in either the basal or calcium-dependent transcription of the involucrin gene.
Keratin-1 gene expression is also known to be up-regulated by extracellular calcium. A region of 422 bp of the human keratin-1 promoter has been shown to contain a calcium-dependent element (19-21). Within this region, there is an AP-1 site and two regions that are homologous to the human involucrin promoter (Figure 6). Since both genes are induced by calcium, one or both of these sites may be involved in mediating the effect of calcium on gene transcription.
The mechanism for calcium-dependent regulation of the CRE is well known and involves CREB and Ca2+/Calmodulin-dependent protein kinase II and IV (5-8). The DNA sequence of the involucrin promoter reveals a putative CRE at -2441. However, deletions of this CRE as in the F-2402 construct did not reduce the calcium-dependent transcription of the reporter gene. Thus, this CRE appears not to be important for the calcium-dependent regulation of the involucrin gene. However, deletions of the region between -2474 and -2402 in this construct did cause a 75% decrease in the basal reporter activity, suggesting that the CRE may play a role in the basal reporter activity.
In conclusion, calcium induces involucrin mRNA transcription in NHK. Calcium can also induce luciferase reporter activity in a construct driven by a 3.7 fragment of the human involucrin gene. This effect is mediated by a calcium-responsive element within a region of the involucrin gene likely to be important for calcium-dependent regulation of the involucrin gene during epidermal differentiation.