Egr-1 expression in surface Ig-mediated B cell activation. Kinetics and association with protein kinase C activation

We have studied the expression of an immediate/early type gene, Egr-1, in murine B lymphocyte responses to Ag receptor-generated signals. The Egr-1 gene encodes a zinc finger protein with sequence-specific DNA binding activity and is believed to act as an intracellular "third messenger," to couple receptor-generated signals to activation-associated changes in gene expression. We show here that Egr-1 mRNA expression is rapidly and transiently (returning to basal levels by 6 h) induced after receptor crosslinking with anti-receptor antibodies. Egr-1 protein expression is more prolonged, maintaining detectable levels through 12 h. The induction of Egr-1 is a primary response to Ag receptor signaling, as it is independent of new protein synthesis and is inhibited by actinomycin D. We have also examined the linkage of Egr-1 to known signaling pathways associated with G0 to G1 transition by these cells in response to signals generated through the B cell Ag receptor. Egr-1 mRNA was not induced after elevation of intracellular free Ca2+. In contrast, the pharmacologic agents PMA and SC-9, which directly activate protein kinase C, both cause marked increases in Egr-1 mRNA levels with the same kinetics as observed after anti-receptor antibody stimulation. Further, the protein kinase C inhibitors H7, sangivamycin, and staurosporin block anti-receptor antibody-induced expression of Egr-1, thus, B cell Ag receptor-linked Egr-1 expression is likely coupled to the protein kinase C component of transmembrane signaling. Preliminary promoter mapping studies are consistent with this conclusion, because both PMA and anti-receptor antibody act through the same or overlapping cis-regulatory elements.     

Egr-1 upregulates OPN through direct binding to its promoter and OPN upregulates Egr-1 via the ERK pathway

Early growth response factor-1 (Egr-1) and osteopontin (OPN) play important roles in the migration and proliferation of vascular smooth muscle cells (VSMC), but little is known about their relationship. Therefore, we transfected VSMCs with either Egr-1 cDNA, Opn cDNA, a DNA enzyme designed to target Egr-1 (ED5), or antisense Opn oligodeoxynucleotides and examined changes in Egr-1 and OPN expression at the mRNA and protein levels. OPN expression levels were increased in cells that were stably transfected with Egr-1 cDNA. By contrast, both Egr-1 and OPN expression were reduced when ED5 was transfected into Egr-1-expressing cells. Similarly, Opn transfection upregulated Egr-1 levels, while Opn anti-sense oligodeoxynucleotide transfection decreased Egr-1 expression. ChIP analysis showed that Egr-1 binds to the Opn gene promoter. Furthermore, treatment with the extracellular-regulated protein kinase (ERK) inhibitor PD98059 inhibited the upregulation of Egr-1 by OPN. We find that Egr-1 and OPN positively regulate each other in VSMCs.