Mitogen-activated Protein Kinase Phosphatase-1 (MKP-1) Expression Is Induced by Low Oxygen Conditions Found in Solid Tumor Microenvironments

Abstract

Pathophysiological hypoxia is an important modula-tor of gene expression in solid tumors and other patho-logic conditions. We observed that transcriptional acti-vation of the c-jun proto-oncogene in hypoxic tumor cells correlates with phosphorylation of the ATF2 tran-scription factor. This finding suggested that hypoxic signals transmitted to c-jun involve protein kinases that target AP-1 complexes (c-Jun and ATF2) that bind to its promoter region. Stress-inducible protein kinases capa-ble of activating c-jun expression include stress-acti-vated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and p38 members of the mitogen-activated protein kinase (MAPK) superfamily of signaling mole-cules. To investigate the potential role of MAPKs in the regulation of c-jun by tumor hypoxia, we focused on the activation SAPK/JNKs in SiHa human squamous carci-noma cells. Here, we describe the transient activation of SAPK/JNKs by tumor-like hypoxia, and the concurrent transcriptional activation of MKP-1, a stress-inducible member of the MAPK phosphatase (MKP) family of dual specificity protein-tyrosine phosphatases. MKP-1 antag-onizes SAPK/JNK activation in response to diverse en-vironmental stresses. Together, these findings identify MKP-1 as a hypoxia-responsive gene and suggest a crit-ical role in the regulation of SAPK/JNK activity in the tumor microenvironment. Tumor hypoxia contributes directly to processes involved in malignant progression, such as angiogenesis (1, 2), the elimi-nation of p53 tumor suppressor activity (3), and genetic insta-bility (4). Recognition that hypoxic tumor microenvironments are important for the development of these phenotypes has stimulated interest in how transformed cells respond to hy-poxic signals (2, 5, 6). Several hypoxia-sensitive mammalian transcription factors have been described, including heat shock transcription factor-1 (HSF-1) (7), c-Fos and c-Jun (5, 8, 9), NF-␬B (10), a retrotransposon-like VL30 element (11), p53 (3), and hypoxia-inducible factor-1 (HIF-1) (12). We reported that the c-jun proto-oncogene is induced at the message and protein levels in hypoxic SiHa human squamous carcinoma cells (5). Further investigation of the mechanism of this induction demonstrated that activation of the c-jun pro-moter by hypoxia correlates with phosphorylation of the trans-activation domain of the ATF2 1 transcription factor (13). Since c-Jun and ATF2 dimers are AP-1 complexes that bind to the c-jun promoter region (14), this finding suggested that hypoxic signals transmitted to the promoter are mediated in part by protein kinases that target both ATF2 and c-Jun. Stress-induc-ible protein kinases capable of activating the c-jun promoter include the SAPK/JNK and p38 MAPK families of the MAPK superfamily of serine/threonine kinases (15, 16). Since both SAPK/JNKs and p38 MAPK are sensitive to redox stresses, such as those associated with ischemia-reperfusion events (17– 20), we investigated the effect of tumor-like hypoxia on their induction in transformed cells. In these studies, which are described in detail below, we observed that both SAPK/JNK and p38 MAPK activities are induced by hypoxia, but the inductions are transient. Because activated SAPK/JNKs and p38 MAPK can be deactivated by members of a family of dual-specificity phosphatases, called MAPK phosphatases (MKPs) (21–23), we hypothesized that the induction of these MAPKs in hypoxic cells is antagonized by redox-responsive members of the MKP family. In particular, we evaluated MKP-1 and -2 as possible contributors to this inhibitory activ-ity, as they are widely expressed immediate-early gene prod-ucts that are induced by a variety of stimuli (23–26). Here, we report that hypoxia transiently induces SAPK/JNK as well as p38 MAPK activity in SiHa cells, and concurrently induces a SAPK/JNK phosphatase activity. This transient in-duction of SAPK/JNK activity correlates with both the tran-scriptional activation of the gene for the MKP family member MKP-1 and the enhanced expression of MKP-1 mRNA. The hypoxia-inducible expression of MKP-1 mRNA is reversible, returning to the aerobic level on reoxygenation. Together, these findings show that MKP-1 is a hypoxia-responsive phosphatase and imply that it contributes to the attenuation of SAPK/JNK activity stimulated in hypoxic cells. In the context of tumor