Thapsigargin Induces Expression of Activating Transcription Factor 3 in Human Keratinocytes Involving Ca2+ Ions and c-Jun N-Terminal Protein Kinase

Abstract

Thapsigargin is a specific inhibitor of the sarco/endoplasmic reticulum Ca 2ϩ ATPase of the endoplasmic reticulum. Here, we show that stimulation of human HaCaT keratinocytes with nanomolar concentrations of thapsigargin triggers expression of activating transcription factor (ATF) 3, a basic-region leucin zipper transcription factor. ATF3 expression was also up-regulated in thapsigargin-stimulated glioma cells, hepatoma cells, retinal pigment epithelial cells, and airway epithelial cells. Thapsigargin-induced up-regulation of ATF3 expression in keratinocytes was attenuated by BAPTA-acetoxymethyl ester or by expression of the Ca 2ϩ -binding protein parvalbumin in the cytosol of HaCaT cells but not by a panel of pharmacological agents that chelate extracellular Ca 2ϩ (EGTA) or inhibit either ryanodine receptors (dantrolene) or voltage-gated Ca 2ϩ channels (nifedipine). Hence, elevated levels of intracellular Ca 2ϩ , released from intracellular stores, are essential for the effect of thapsigargin on the biosynthesis of ATF3. The thapsigargininduced signaling pathway was blocked by expression of either mitogen-activated protein kinase phosphatase-1 or -5. Experiments involving pharmacological and genetic tools revealed the importance of c-Jun N-terminal protein kinase (JNK) within the signaling cascade, whereas inhibition of extracellular signal-regulated protein kinase or p38 protein kinase did not attenuate thapsigargin-induced expression of ATF3. Functional studies showed that treatment of HaCaT keratinocytes with thapsigargin led to a 2-fold induction of caspase-3/7 activity. The up-regulation of caspase-3/7 activity in thapsigargin-stimulated HaCaT cells was attenuated by inhibition of JNK. Together, these data show that stimulation of HaCaT cells with thapsigargin induces a specific signaling pathway in keratinocytes involving activation of JNK, biosynthesis of ATF3, and up-regulation of caspase-3/7 activity.

Transient Receptor Potential Melastatin-3 (TRPM3)–Induced Activation of AP-1 Requires Ca2+ Ions and the Transcription Factors c-Jun, ATF2, and Ternary Complex Factor

“…The lentiviral transfer vectors pFUW-REST/Elk-1DC, pFUW-MEKK1D, pFUW-MKK6E, pFUWc-JunDN, pFUW-ATF2DN, and pFUW-MKP-1 have been described previously (Mayer et al, 2008Rössler and Thiel, 2009;Müller et al, 2010;Spohn et al, 2010;Thiel et al, 2012). The GAL4 expression plasmid pFA2ATF2 was purchased from Stratagene (Amsterdam, The Netherlands).…”

“…Proteins were separated by SDS-PAGE, blotted, and incubated with antibodies directed against c-Jun (Santa Cruz, Heidelberg, Germany) or ATF2 (Santa Cruz). The antibody directed against histone deacetylase-1 was used as a loading control as previously described (Spohn et al, 2010;Mayer et al, 2011). To detect FLAG-tagged proteins, we used the M2 monoclonal antibody directed against the FLAG epitope (Sigma-Aldrich, Steinheim, Germany) at 1:3000 dilution.…”

“…To detect FLAG-tagged proteins, we used the M2 monoclonal antibody directed against the FLAG epitope (Sigma-Aldrich, Steinheim, Germany) at 1:3000 dilution. Immunoreactive bands were detected via enhanced chemiluminescence as described (Spohn et al, 2010;Mayer et al, 2011). Values are expressed as the mean 6 S.D.…”

Temporins A and B Stimulate Migration of HaCaT Keratinocytes and Kill Intracellular Staphylococcus aureus

“…After different time periods (30 min and 3, 6, 9, and 12 h), cells were washed with PBS and fixed with 3.7% formaldehyde for 10 min at ϩ4°C. Afterwards, they were washed with PBS, treated with 0.1% Triton X-100 for 10 min, and stained with 2 g/ml of Hoechst 33258 for 10 min at room temperature (45). The coverslips were placed on a glass slide with buffered glycerol and visualized under a fluorescence confocal microscope (Olympus FV1000).…”

Signal Transduction of Pregnenolone Sulfate in Insulinoma Cells

“…Proteins were separated by SDS-PAGE, blotted, and incubated with antibodies directed against Egr-1 (Santa Cruz, Heidelberg, Germany, sc-189), HDAC-1 (Upstate Biotechnology, Lake Placid, NY, 05-100), TRPM3 (12), Calnexin (Stressgen), or Synapsin I (a kind gift of T. C. Südhof, Stanford University). The antibody directed against histone deacetylase-1 (HDAC1) was used as a loading control as previously described (36). To detect FLAGtagged proteins, we used the M2 monoclonal antibody directed against the FLAG epitope (Sigma, number F3165) at 1:3000 dilution.…”