Abstract
The small proline-rich protein gene (spr1) is a marker whose expression is frequently associated with squamous cell differentiation. We observed that the expression of the spr1 gene is strongly induced by phorbol 12-myristate 13-acetate (PMA). Both the time course result and the nuclear run-on transcriptional assay suggested that the regulation of spr1 expression by PMA is controlled at the transcriptional level. To understand the nature of this regulation, human genomic clones of the spr1 gene were isolated. DNA sequence analysis revealed that the human spr1 gene contains two exons and a single intron located within the 5'-untranslated region. An AP-1 binding site (TGAGTCA) is found at -142, and a putative cyclic AMP-responsive element (TGAGGTCA) at -597 base pairs upstream of the transcription start site. A chimeric construct containing the 5'-flanking region of the spr1 gene and the chloramphenicol acetyltransferase (CAT) reporter gene was used to transfect HeLa cells or monkey primary TBE cells. The CAT activity in transfected cells is stimulated 7.5-11-fold by PMA, and the stimulation is inhibited by a protein kinase C inhibitor or by pretreating cells with PMA to down-regulate the protein kinase C activity. The CAT activity is also stimulated 3.5-fold by dibutyryl cyclic AMP, a protein kinase A activator. The stimulations by PMA and cAMP are additive. These results suggest that protein kinase C and probably protein kinase A play important roles in regulating the transcription of the spr1 gene.
Highlights
From the icalifornia PrimateResearch Center and the $Department of Biochemistry and Biophysics, University of California, Dauis, Californh 95616
DNA sequence analysis revealed that the human sprl gene contains two exons anda single intron located within that the spr1 message is often found in abundance in tissues containing squamous epithelium, such astongue, esophagus, and skin [3]
P M A and Dibutyryl CAMP Stimulate the Expressioonf the sprl Gene-We demonstrated previously that thesprl mRNA level in TBE cells is elevated by PMA [3]
Summary
The "P-labeled monkey sprl cDNA probe [3]was added to the prehybridizationsolution at a concentrationgreaterthan 1 X lo cpm/ml. The cells were scraped from culture dishes and centrifuged a t 500 X g for 5 min at 4 "C. Northern blot filterwas prepared and hybridized to "'P-labeled mon- phage plaqueswere plated (50,000/150-mm dish) andwere lifted onto key sprl cDNA and 18 S ribosomal probes as indicated. '"P-Labeled monkey sprl from cultures without any treatment; lane, treated with cyclohexi- cDNAprobe [3]prepared by therandomprimer labeling k i t was mide (5 pg/ml); lane 3, treated with dibutyryl cAMP (1 mM); lane 4, hybridized tothefilters according tothescreening procedure of treated with dibutyryl cAMP andcycloheximide; lane 5,treated with Maniatis et al [18].Two to three rounds of rescreening were per-. PMA (100 ng/ml);lane 6, treatedwithPMAand cycloheximide. formed until pure and specific clones were obtained
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
