Caspase-dependent proteolytic cleavage of STAT3alpha in ES cells, in mammary glands undergoing forced involution and in breast cancer cell lines

Abstract

Background: The STAT (Signal Transducers and Activators of Transcription) transcription factor family mediates cellular responses to a wide range of cytokines. Activated STATs (particularly STAT3) are found in a range of cancers. Further, STAT3 has anti-apoptotic functions in a range of tumour cell lines. After observing a proteolytic cleavage in STAT3alpha close to a potential apoptotic caspase protease cleavage site we investigated whether STAT3alpha might be a caspase substrate.

Methods: STAT3alpha status was investigated in vitro in several cell systems:- HM-1 murine embryonic stem (ES) cells following various interventions; IOUD2 murine ES cells following induction to differentiate along neural or adipocyte lineages; and in a number of breast cancer cell lines. STAT3alpha status was also analysed in vivo in wild type murine mammary glands undergoing controlled, forced involution.

Results: Immunoblotting for STAT3alpha in HM-1 ES cell extracts detected amino and carboxy terminal species of approximately 48 kDa and 43 kDa respectively--which could be diminished dose-dependently by cell treatment with the nitric oxide (NO) donor drug sodium nitroprusside (SNP). UV irradiation of HM-1 ES cells triggered the STAT3alpha cleavage (close to a potential caspase protease cleavage site). Interestingly, the pan-caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone (z-VAD-FMK) and the JAK2 tyrosine kinase inhibitor AG490 both inhibited cleavage dose-dependently, and cleavage was significantly lower in a heterozygous JAK2 knockout ES cell clone. STAT3alpha cleavage also occurred in vivo in normal murine mammary glands undergoing forced involution, coinciding with a pulse of phosphorylation of residue Y705 on full-length STAT3alpha. Cleavage also occurred during IOUD2 ES cell differentiation (most strikingly along the neural lineage) and in several human breast cancer cell lines, correlating strongly with Y705 phosphorylation.

Conclusion: This study documents a proteolytic cleavage of STAT3alpha into 48 kDa amino and 43 kDa carboxyl terminal fragments in a range of cell types. STAT3alpha cleavage occurs close to a potential caspase site, and can be inhibited dose-dependently by SNP, AG490 and z-VAD-FMK. The cleavage seems to be caspase-dependent and requires the phosphorylation of STAT3alpha at the Y705 residue. This highly regulated STAT3alpha cleavage may play an important role in modulating STAT3 transcriptional activity.

Figure 1

Proteolytic cleavage of STAT3α in HM-1 murine ES cells to 48 kDa and 43 kDa fragments can be inhibited by the NO-donor SNP. A. An antibody (Santa Cruz sc-482) directed against amino acids 750–769 at the carboxy terminus of STAT3α revealed full-length STAT3α and a 43 kDa carboxy terminal cleavage product. 90 μg of protein were loaded per lane. B. Blot stripped and re-probed with an antibody (Cell Signaling Technology #9132) that recognises an epitope around the STAT3 Y705 residue within the carboxy terminal region, again detecting the 43 kDa cleavage fragment. C. Blot stripped and re-probed with an antibody that recognises an epitope within the STAT3 amino terminal amino acid 1–175 region (BD/Transduction Laboratories S21320/610190), detecting full-length STAT3α and a STAT3-derived 48 kDa amino terminal cleavage fragment. Cell culture treatment with the NO-donor (and caspase inhibitor) SNP for 2 hours reduced the level of the 43 kDa and 48 kDa fragments in a dose-dependent manner (lanes 1–3, 6–8 in panels A-C) in HM-1 ES cells cultured in either the presence (+LIF, lanes 1–5) or absence (-LIF, lanes 6y10) of added LIF in the medium. In contrast, the proteasome inhibitor MG132 added to 10 μM in the medium for 2 hours (with DMSO vehicle to a final concentration of 0.2% vol/vol) could not inhibit cleavage (lanes 4, 9 in A-C) relative to untreated cells, or DMSO vehicle alone (D). D. Blot stripped and re-probed with an anti β-Actin monoclonal antibody (Sigma, AC-74) to confirm equal amounts of protein loaded per lane.

Figure 2

Modulation of STAT3α cleavage by caspase and JAK2 protein tyrosine kinase activity. A. Immunoblot showing a significant increase in STAT3α cleavage following UV irradiation. The Santa Cruz sc-482 antibody (recognising the carboxy terminus of STAT3α) revealed a significant increase in the level of the 43 kDa fragment following UV irradiation (+UV) of HM-1 ES cells (254 nm, 1 mJ/cm², with harvesting 13 hours later) compared to control un-irradiated (-UV) cells in the presence (D) or absence (-) of 0.5% vol/vol DMSO vehicle, 100 μg of whole cell extract protein loaded per lane. B. Immunoblot demonstrating dose-dependent inhibition of STAT3α cleavage by the pan-caspase inhibitor z-VAD-FMK and the JAK2 tyrosine kinase inhibitor AG490. The 43 and 48 kDa STAT3α fragments were essentially abolished by pretreating HM-1 ES cells with z-VAD-FMK to 60 μM (lanes 1, 2, 6–8, C-term. STAT3α and N-term. STAT3 panels) or AG490 to 250 μM (lanes 1–5, C-term. STAT3α and N-term. STAT3 panels) for 18 hours in the cell culture medium before harvesting. The 250 μM AG490-mediated abolition of STAT3α cleavage correlated with loss of tyrosine phosphorylation of the STAT3α Y705 residue (lane 5, PY 705 STAT3 panel). 75 μg of whole cell extract protein loaded per lane, DMSO added to 0.5% vol/vol in the culture medium in the z-VAD-FMK and AG490 treatments and the DMSO control (D). C. Identical behaviour of HM-1 ES cell cytoplasmic and nuclear extracts following AG490-mediated inhibition of STAT3α cleavage and inability of 50 μM MG132 to inhibit STAT3α cleavage. 100 μg of cytoplasmic or nuclear extract protein loaded per lane, DMSO added to 1% vol/vol in the culture medium for AG490 and MG132 treatments and DMSO controls (D), treatments given for 7 hours prior to cell harvesting. Immunoblots used the Santa Cruz sc-482 antibody. D. Down-regulation of STAT3α cleavage in JAK2 heterozygous knockout (JAK2 +/-) ES cells. Wild type HM-1 and JAK2 +/- ES cells were cultured with (+LIF) or without (-LIF) added LIF for 3 days prior to harvesting. Cytoplasmic extracts (100 μg protein per lane) were immunoblotted with the Santa Cruz sc-482 antibody. E. Immunoblots of RIPA extracts of wild type (W.T.) and SMAD4 transgenic (+) murine mammary glands from day 10 lactation (D10L) and 2 day (D2In.), 3 day (D3In.) and 6 day (D6In.) forced involution timepoints showing the close linkage between appearance of tyrosine 705 phosphorylated STAT3α and the generation of the 43 kDa carboxy terminal STAT3α cleavage product. 20 μg of RIPA whole cell extract protein was loaded per lane.

Figure 3

STAT3 cleavage during ES cell differentiation along neural or adipocyte cell lineages. A. Short term differentiation of murine IOUD2 ES cells following removal of LIF for 48 hours. In the adipocyte differentiation protocol, cells were treated with RA between 48 and 72 hours, while cells differentiating along the neural lineage were untreated. Whole cell extracts were immunoblotted with either the anti-STAT3 amino terminal antibody (upper panel, 48 kDa cleaved fragment) or the anti-STAT3 PY705 antibody (lower panel, 43 kDa cleaved fragment). B. 20 day differentiation time course of IOUD2 ES cells along the neural cell lineage. Cells were harvested at the indicated timepoints and whole cell extracts immunoblotted with either the anti-STAT3 amino terminal antibody (upper panel, 48 kDa cleaved fragment) or the anti-STAT3 PY705 antibody (lower panel, 43 kDa cleaved fragment). C. 20 day differentiation time course of IOUD2 ES cells along the adipocyte cell lineage. Cells were harvested at the indicated timepoints and whole cell extracts immunoblotted with either the anti-STAT3 amino terminal antibody (upper panel, 48 kDa cleaved fragment) or the anti-STAT3 PY705 antibody (lower panel, 43 kDa cleaved fragment). Arrows indicate cleaved fragments and the asterisk non-specific bands.

Figure 4

Cleavage of STAT3α in human breast cancer cell lines. Whole cell extracts from one normal human breast cell line (HB4A) and 7 breast cancer cell lines with varying levels of activated, tyrosine phosphorylated, STAT3α were immunoblotted with either the anti-STAT3 amino terminal antibody (upper panel, 48 kDa cleaved fragment) or the anti-STAT3 PY705 antibody (lower panel, 43 kDa cleaved fragment). Over exposure of the upper blot revealed an additional cleavage product of approximately 25 kDa that was most apparent in the GI-101 cell line which has the highest level of tyrosine phosphorylated STAT3α in the breast cancer cell lines examined. Extracts from C57Bl/6 mouse mammary glands were loaded as controls: 5d Gest = day 5 pregnancy; 10d Lac = 10 days of lactation; 96 Hr In = 96 hours following forced weaning at day 10 lactation. Arrows indicate cleaved fragments.