Role of p38 in stress activation of Sp1

Abstract

Cell stressors such as physical forces can activate Sp1-dependent genes but the regulatory mechanisms are not defined. We determined if the stress-induced MAP kinase, p38, can phosphorylate Sp1 and thereby regulate the Sp1 target gene FLNA. We used Rat-2 cells and human gingival fibroblasts to examine stress-induced activation of an Sp1-dependent gene and SL2 cells, an Sp1-deficient model system, to facilitate interaction studies of transfected Sp1 with regulatory factors. Mechanical stress applied to Rat-2 cells increased promoter activity of the Sp1 target gene filamin A by > 5-fold; activation was blocked by mutations to Sp1 binding sites in the filamin A promoter. Transfection experiments in SL2 cells with Sp1 expression vectors showed that when co-transfected with constitutively active p38, wild-type Sp1 but not an Sp1 binding mutant, increased promoter activity of the Sp1 target gene, filamin A, and enhanced binding of nuclear extracts to a filamin A promoter oligonucleotide. Filamin A promoter activity was blocked by dominant negative p38. Sp1 that was phosphorylated at Thr⁴⁵³ and Thr⁷³⁹ by constitutively active p38 bound to the filamin A promoter more effectively than un-phosphorylated Sp1. Recombinant active p38 phosphorylated wild-type Sp1 in vitro while the Sp1 Thr⁴⁵³Thr⁷³⁹ double mutant protein showed > 3-fold reduction of phosphorylation. We conclude that stress activation of p38 phosphorylates Sp1 at specific threonine residues, modifications which in turn enhance the expression of Sp1-dependent genes.

Introduction

Cells respond to physical stressors such as extracellular forces by activating signaling pathways that regulate cytoprotective genes (Chien et al., 1998, DeMali et al., 2003, Shyy and Chien, 2002). An early step in the stress response includes phosphorylation of the MAP kinase p38 (D'Addario et al., 2001) but the functional relationship between p38 phosphorylation and activation of cytoprotective genes is poorly understood. The Sp1 transcription factor can be activated by physical forces (D'Addario et al., 2001) and has been implicated in shear force-induced regulation of the membrane-type matrix metalloproteinase 1 gene (Yun et al., 2002) and the FLNA gene (D'Addario et al., 2001). Notably, FLNA is a mechanical stress-sensitive gene that has been linked to cytoprotective responses to tensile forces (Kainulainen et al., 2002). Filamin A protects cells against force-induced death likely by stabilizing the plasma membrane and preventing irreversible membrane depolarization. The FLNA promoter contains Sp1 binding sites that are mechanically activated (D'Addario et al., 2002) and thus Sp1 may be a stress-sensitive transcription factor.

Sp1 was one of the first eukaryotic transcription factors to be described and cloned (Dynan and Tjian, 1983a, Dynan and Tjian, 1983b). Sp1 and Sp family members are differentially acetylated, phosphorylated and/or glycosylated, and bind variants of a GC-rich box. They were first characterized by their ability to activate SV40 viral gene expression (Bouwman et al., 2000, Suske, 1999). The Sp family comprises at least 8 members that show some measure of specificity and redundancy, although Sp1 knockout mice are unique in exhibiting embryonic lethality (Marin et al., 1997). Sp1 co-operatively interacts with other transcription factors and is activated by phosphorylation (Fojas de Borja et al., 2001, Milanini-Mongiat et al., 2002).

While it is known that certain mechanical stressors can selectively activate p38 and not JNK or ERK (Wang et al., 2003), and promote the expression of Sp1-dependent genes (D'Addario et al., 2001), the nature of the interaction of p38 with Sp1, and the stress activation mechanism of Sp1, are not defined. Accordingly we assessed the role of p38 in regulating FLNA. We show here that Sp1 is phosphorylated in vitro on threonine 453 and 739 by p38 and that mutation of these residues abrogates transcriptional activation of FLNA.