p38δ genetic ablation protects female mice from anthracycline cardiotoxicity.

The efficacy of an anthracycline antibiotic doxorubicin (DOX) as a chemotherapeutic agent is limited by dose-dependent cardiotoxicity. DOX is associated with activation of intracellular stress signaling pathways including p38 MAPKs. While previous studies have implicated p38 MAPK signaling in DOX-induced cardiac injury, the roles of the individual p38 isoforms, specifically, of the alternative isoforms p38γ and p38δ, remain uncharacterized. We aimed to determine the potential cardioprotective effects of p38γ and p38δ genetic deletion in mice subjected to acute DOX treatment. Male and female wild-type (WT), p38γ-/-, p38δ-/-, and p38γ-/-δ-/- mice were injected with 30 mg/kg DOX and their survival was tracked for 10 days. During this period, cardiac function was assessed by echocardiography and electrocardiography and fibrosis by Picro Sirius Red staining. Immunoblotting was performed to assess the expression of signaling proteins and markers linked to autophagy. Significantly improved survival was observed in p38δ-/- female mice post-DOX relative to WT females, but not in p38γ-/- or p38γ-/-δ-/- male or female mice. The improved survival in DOX-treated p38δ-/- females was associated with decreased fibrosis, increased cardiac output and LV diameter relative to DOX-treated WT females, and similar to saline-treated controls. Structural and echocardiographic parameters were either unchanged or worsened in all other groups. Increased autophagy, as suggested by increased LC3-II level, and decreased mammalian target of rapamycin activation was also observed in DOX-treated p38δ-/- females. p38δ plays a crucial role in promoting DOX-induced cardiotoxicity in female mice by inhibiting autophagy. Therefore, p38δ targeting could be a potential cardioprotective strategy in anthracycline chemotherapy.NEW & NOTEWORTHY This study for the first time identifies the sex-specific roles of the alternative p38γ and p38δ MAPK isoforms in promoting doxorubicin (DOX) cardiotoxicity. We show that p38δ and p38γ/δ systemic deletion was cardioprotective in female but not in male mice. Cardiac structure and function were preserved in DOX-treated p38δ-/- females and autophagy marker was increased.

Myeloid cell deficiency of p38γ/p38δ protects against candidiasis and regulates antifungal immunity.

Candida albicans is a frequent aetiologic agent of sepsis associated with high mortality in immunocompromised patients. Developing new antifungal therapies is a medical need due to the low efficiency and resistance to current antifungal drugs. Here, we show that p38γ and p38δ regulate the innate immune response to C. albicans We describe a new TAK1-TPL2-MKK1-ERK1/2 pathway in macrophages, which is activated by Dectin-1 engagement and positively regulated by p38γ/p38δ. In mice, p38γ/p38δ deficiency protects against C. albicans infection by increasing ROS and iNOS production and thus the antifungal capacity of neutrophils and macrophages, and by decreasing the hyper-inflammation that leads to severe host damage. Leucocyte recruitment to infected kidneys and production of inflammatory mediators are decreased in p38γ/δ-null mice, reducing septic shock. p38γ/p38δ in myeloid cells are critical for this effect. Moreover, pharmacological inhibition of p38γ/p38δ in mice reduces fungal burden, revealing that these p38MAPKs may be therapeutic targets for treating C. albicans infection in humans.

Combined deletion of p38γ and p38δ reduces skin inflammation and protects from carcinogenesis.

The contribution of chronic skin inflammation to the development of squamous cell carcinoma (SCC) is poorly understood. While the mitogen-activated protein kinase p38α regulates inflammatory responses and tumour development, little is known about the role of p38γ and p38δ in these processes. Here we show that combined p38γ and p38δ (p38γ/δ) deletion blocked skin tumour development in a chemically induced carcinogenesis model. p38γ/δ deletion reduced TPA-induced epidermal hyperproliferation and inflammation; it inhibited expression of proinflammatory cytokines and chemokines in keratinocytes in vitro and in whole skin in vivo, resulting in decreased neutrophil recruitment to skin. Our data indicate that p38γ/δ in keratinocytes promote carcinogenesis by enabling formation of a proinflammatory microenvironment that fosters epidermal hyperproliferation and tumourigenesis. These findings provide genetic evidence that p38γ and p38δ have essential roles in skin tumour development, and suggest that targeting inflammation through p38γ/δ offers a therapeutic strategy for SCC treatment and prevention.

Alternative p38 MAPKs are essential for collagen-induced arthritis.

OBJECTIVE: The role of most p38 MAPK isoforms in inflammatory arthritis is not known. This study was undertaken to evaluate p38γ and p38δ deficiency in the collagen-induced arthritis (CIA) model. METHODS: Wild-type, p38γ(-/-) , p38δ(-/-) , and p38γ/δ(-/-) mice were immunized with chicken type II collagen, and disease activity was evaluated by semiquantitative scoring and histologic assessment. Serum cytokine levels and in vitro T cell cytokine responses were quantified by flow cytometry and multiplex analysis, and serum anticollagen antibody levels by enzyme-linked immunosorbent assay. Cytokine and p38 MAPK isoform expression in joints were determined by quantitative polymerase chain reaction. RESULTS: Compound p38γ and p38δ deficiency markedly reduced arthritis severity compared with that in wild-type mice, whereas lack of either p38γ or p38δ had an intermediate effect. Joint damage was minimal in arthritic p38γ/δ(-/-) mice compared with wild-type mice. The p38γ/δ(-/-) mice had lower levels of pathogenic anticollagen antibodies and interleukin-1ß (IL-1ß) and tumor necrosis factor α than controls. In vitro T cell assays showed reduced proliferation, interferon-γ (IFNγ) production, and IL-17 production by lymph node cells from p38γ/δ(-/-) mice. IL-17 and IFNγ messenger RNA expression in joints was significantly inhibited in p38γ/δ(-/-) mice. Wild-type chimeric mice with p38γ/δ(-/-) bone marrow did not show decreased CIA. CONCLUSION: Reduced disease severity in p38γ/δ(-/-) mice was associated with lower cytokine production and anticollagen antibody responses than in controls, indicating that p38γ and p38δ are crucial regulators of inflammatory joint destruction in CIA. Our findings indicate that p38γ and p38δ are potential therapeutic targets in complex diseases, such as rheumatoid arthritis, that involve innate and adaptive immune responses.

Role of p38 mitogen-activated protein kinase isoforms in murine skin inflammation induced by 12-O-tetradecanoylphorbol 13-acetate.

p38 mitogen-activated protein kinase plays a pivotal role in skin inflammation. The purpose of this study was to investigate the role of the various p38 isoforms. p38ß/δ-knockout-C57BL/6 mice were generated, studied in a 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced skin inflammation model and compared with wild-type mice. The inflammatory response was determined by ear thickness, myeloperoxidase activity and histology. mRNA and protein expression of interleukin (IL)-1ß and IL-6 was determined by quantitative real-time reverse transcription PCR and enzyme-linked immunoassay. In both groups application of TPA resulted in a significant increase in inflammation, and pretreatment with the p38α/ß inhibitor, SB202190 resulted in a significant inhibition. A significantly slower onset but prolonged duration of the response was seen in p38ß/δ knockout mice. This was paralleled by a significant, but transient, lower IL-1ß and IL-6 protein expression in p38ß/δ knockout mice. Although the p38α isoform is important, our data also demonstrate an important role of the p38ß and/or δ isoforms in the regulation of TPA-induced skin inflammation.

Mouse embryonic stem cells lacking p38alpha and p38delta can differentiate to endothelial cells, smooth muscle cells, and epithelial cells.

The p38 mitogen-activated protein (MAP) kinases (p38) are important signaling molecules that regulate various cellular processes. Four isoforms of p38 family, p38alpha, p38beta, p38gamma, and p38delta, have been identified in mammalian cells. Previous studies have shown that p38alpha knockout is embryonic lethal in mice. At the cellular level, p38alpha is abundantly expressed in mouse embryonic stem cells (ESCs), but p38alpha knockout (p38alpha-/-) ESCs can differentiate to endothelial cells (ECs), smooth muscle cells (SMCs), and neurons. We speculate that the lost function of p38alpha in p38alpha-/- ESCs may be compensated for by the redundant function of other isoforms. To test this hypothesis, we used siRNA approach to knock down the expression of p38delta, the second abundant isoform in ESCs. ESCs stably expressing p38delta siRNA were established from p38alpha-/- ESCs, resulting in 80% reduction of p38delta mRNA expression. However, these ESCs, deficient of both p38alpha and p38delta, could still differentiate into ECs and SMCs. We extended our investigation to test if these cells can differentiate into epithelial cells in which p38delta has been shown to regulate epidermis differentiation. Our results demonstrate again that ESC differentiation to epithelial cells is independent of p38alpha and p38delta. We conclude that p38alpha and p38delta are not essential for ESC differentiating into ECs, SMCs, or epithelial cells although numerous studies have shown that the two kinases regulate various cellular activities in aforementioned cells. Our results highlight the possibility that p38 MAP kinases may play less significant roles in ESC differentiation than in the regulation of cellular activities of fully differentiated somatic cells.

Efficient adult skeletal muscle regeneration in mice deficient in p38beta, p38gamma and p38delta MAP kinases.

Adult skeletal muscle is a very stable tissue containing a small population of myofiber-associated quiescent satellite cells compared with late embryonic/neonatal skeletal muscle, which contains highly proliferating myoblasts and small actively growing myofibers, suggesting that specific regulatory pathways may control myogenesis at distinct developmental stages. The p38 MAPK signaling pathway is central for myogenesis, based on studies using immortalized and neonatal primary myoblasts in vitro. However, the contribution of this pathway to adult myogenesis has never been investigated. Four p38 isoforms (p38alpha, p38beta, p38gamma and p38delta) exist in mammalian cells, being p38alpha and p38gamma the most abundantly expressed isoforms in adult skeletal muscle. Given the embryonic/neonatal lethality of p38alpha-deficient mice, here we investigate the relative contribution of p38beta, p38gamma and p38delta to adult myogenesis. Regeneration and myofiber growth of adult muscle proceeds with similar efficiency in mice lacking p38beta, p38gamma and p38delta as in wild-type control mice. In agreement with this, there is no difference in adult primary myoblasts behavior in vitro among the different genotypes. Importantly, the pattern of p38 activation (ascribed to p38alpha) remains unperturbed during satellite cell-mediated myogenesis in vitro and adult muscle regeneration in wild type and p38beta-, p38gamma- and p38delta-deficient mice, rendering p38alpha as the essential p38 isoform sustaining adult myogenesis. This study constitutes the first analysis addressing the functionality of p38beta, p38gamma and p38delta in satellite cell-dependent adult muscle regeneration and growth.