Functional cooperation between ASK1 and p21Waf1/Cip1 in the balance of cell-cycle arrest, cell death and tumorigenesis of stressed keratinocytes

Abstract

Both CDKN1A (p21 ^Waf1/Cip1) and Apoptosis signal-regulating kinase 1 (ASK1) play important roles in tumorigenesis. The role of p21 ^Waf1/Cip1 in attenuating ASK1-induced apoptosis by various stress conditions is well established. However, how ASK1 and p21 ^Waf1/Cip1 functionally interact during tumorigenesis is still unclear. To address this aspect, we crossed ASK1 knockout (ASK1KO) mice with p21 ^Waf1/Cip1 knockout (p21KO) mice to compare single and double-mutant mice. We observed that deletion of p21 ^Waf1/Cip1 leads to increased keratinocyte proliferation but also increased cell death. This is mechanistically linked to the ASK1 axis-induced apoptosis, including p38 and PARP. Indeed, deletion of ASK1 does not alter the proliferation but decreases the apoptosis of p21KO keratinocytes. To analyze as this interaction might affect skin carcinogenesis, we investigated the response of ASK1KO and p21KO mice to DMBA/TPA-induced tumorigenesis. Here we show that while endogenous ASK1 is dispensable for skin homeostasis, ASK1KO mice are resistant to DMBA/TPA-induced tumorigenesis. However, we found that epidermis lacking both p21 and ASK1 reacquires increased sensitivity to DMBA/TPA-induced tumorigenesis. We demonstrate that apoptosis and cell-cycle progression in p21KO keratinocytes are uncoupled in the absence of ASK1. These data support the model that a critical event ensuring the balance between cell death, cell-cycle arrest, and successful divisions in keratinocytes during stress conditions is the p21-dependent ASK1 inactivation.

Fig. 1. Inactivation of both p21 and ASK1 has not a major impact on differentiation of keratinocytes.

A Primary mouse keratinocytes were established from 2-day-old newborn mice and maintained in low calcium medium. Cells were analyzed for western blot analysis with the indicated antibodies 5 days post-seeding. B H&Es taining of single p21KO, ASK1KO, ASK1/p21KO, and wild-type (WT) skin at birth (2-day-old newborn mice). 10X microscope field (scale bar: 100 μm). C Equal amounts of protein extract from WT, p21KO, ASK1KO ASK1/p21KO neonate dorsal skin (2-day-old newborn mice) were separated by SDS-PAGE and analyzed by western blot with antibodies directed against keratin 1 and loricrin. Vinculin has been used as a loading control (D) Immunohistochemical labeling of dorsal skin sections from 2-day-old newborn WT, p21KO, ASK1KO ASK1/p21KO mice using antibodies specific to loricrin shows a similar expression level and localization of the differentiation marker in WT and KO mice. 20X microscope field (scale bar: 100 μm).

Fig. 2. Increased apoptosis but not increased proliferation of p21-deleted primary keratinocytes is ASK1-dependent.

A Primary mouse keratinocytes were established from 2-day-old newborn mice and maintained in low calcium medium. Cells were analyzed 5 days post-seeding. Cultured keratinocytes cells were pulse labeled with BrdU for 12 h, harvested and prepared for FACS analysis as described in Materials and methods. B Primary mouse keratinocytes were established from 2-day-old newborn mice and maintained in low calcium medium. Cells were seeded in triplicate at a density of 4 × 10⁴ cells/cm² in 6-well culture dishes and analyzed 10 days post-seeding. Once the cells were trypsinized approximately 0.2 mL of the cells were mixed with an equal volume of 0.4% Trypan blue solution and viable cells were counted using a hemocytometer. Results are expressed as mean ± SEM of triplicate experiments. C Flow cytometric analysis of the cell death phenotype by PI staining, the % of apoptotic cells is indicated. Results are expressed as mean ± SEM of triplicate experiments.

Fig. 3. ASK1-p38 axis promotes apoptosis in p21-deficient primary keratinocytes.

A–E Primary mouse keratinocytes were established from 2-day-old newborn mice and maintained in low calcium medium. Cells were analyzed 5 days post-seeding by western blot analysis with the indicated antibodies.

Fig. 4. Inactivation of both p21 and ASK1 has a major impact on DMBA/TPA-induced carcinogenesis.

A In vivo DMBA/TPA induces skin carcinogenesis in 7–9 week old mice. Upper panel the indicated mice were treated once with DMBA (100 μg in 200 μl of acetone) and then continually treated with TPA (10 μg in 200 μl of acetone) twice a week for 20 weeks. The average (8 mice each group) number of papillomas per mouse is shown. The significance of the differences was calculated using one-way ANOVA. *P < 0.05, ***P < 0.0005 are the significance of indicated mice compared with WT mice. B Lower panel, a representative picture of the treated mice.

Fig. 5. Model.

When exposed to environmental stress or aging cultured primary mouse keratinocytes undergo a growth arrest triggered by p21. However, under stress conditions p21 counteracts ASK1-dependent apoptosis. In contrast to wild-type keratinocytes, when exposed to stress p21-deficiency impedes the block of DNA synthesis. Thus, the most severely affected p21-deficient cells are eliminated in the course of stress by ASK1-activation. We propose that ASK1 and p21 function as a checkpoint-tumor surveillance mechanism in which ASK1 represents a mechanism for compensating loss of p21 activity. Thus, loss of the p21/ASK1 axis impairs tumor surveillance with increasing the risk of tumorigenesis.