Cellular senescence, a state of irreversible growth arrest, is implicated in various age-related pathologies, including skin aging. In this study, we investigated the role of CLCA2, a calcium-activated chloride channel accessory protein, in cellular senescence and its implications for skin aging. Utilizing UVB and Nutlin3a-induced senescence models, we observed the upregulation of CLCA2 at both transcriptomic and proteomic levels, suggesting its involvement in senescence pathways. Further analysis revealed that the depletion of CLCA2 led to accelerated senescence onset, characterized by classic senescence markers and a unique secretome profile. In 3D skin equivalent models, SEs constructed with CLCA2 knockdown fibroblasts exhibited features reminiscent of aged skin, underscoring the importance of CLCA2 in maintaining skin homeostasis. Our findings highlight CLCA2 as a novel regulator of cellular senescence and its potential implications for skin aging mechanisms.

OBJECTIVE: Recent studies reported therapeutic effects of monotherapy with either tumour suppressor p53 (p53) agonist or hypoxia-inducible factor 2α (HIF-2α) antagonist for pulmonary hypertension (PH). This study investigated whether a combined treatment of p53 agonist, Nutlin3a, and HIF-2α antagonist, PT2385, would be more effective than monotherapy, based on the cell type-divergent regulation of p53 in pulmonary arterial smooth muscle cells (PASMC) and endothelial cells (PAEC) in patients and animals with PH.
METHODS: The SU5416/hypoxia-induced PH (SuHx-PH) rat model was used, along with cultured human PASMC and PAEC. Western blot, RT-PCR, siRNA and immunohistochemical methods were used along with echocardiography and studies with isolated pulmonary arteries.
RESULTS: Hypoxia-induced proliferation of PASMC is associated with decreased p53, whereas hypoxia-induced PAEC apoptosis is associated with increased p53, via a HIF-2α-dependent mechanism. Combined treatment with Nutlin3a and PT2385 is more effective by simultaneously inhibiting the hypoxia-induced PASMC proliferation and PAEC apoptosis, overcoming the side-effects of monotherapy. These are (i) Nutlin3a exacerbates hypoxia-induced PAEC apoptosis by inducing p53 in PAEC and (ii) PT2385 inhibits PAEC apoptosis because HIF-2α is predominantly expressed in PAEC but lacks direct effects on the hypoxia-induced PASMC proliferation. In rats, combination treatment is more effective than monotherapy in reversing established SuHx-PH, especially in protecting pulmonary arterial vasculature, by normalizing smooth muscle thickening, protecting against endothelial damage and improving function.
CONCLUSIONS: Combination treatment confers greater therapeutic efficacy against PH through a selective modulation of p53 and HIF-2α in PASMC and PAEC.

Escape from apoptosis, one of the characteristic features of cancer cells, is a case that reduces the therapeutic efficacy of apoptosis-inducing molecules used in the cancer treatment. Stabilization of the P53 protein, which is responsible for the regulation of apoptosis mechanism in the cell, is therefore an important therapeutic goal. Nutlin3a inhibits the degradation of the P53 protein, triggers P53-mediated apoptosis in cancer cells and enhances the effectiveness of chemotherapeutics. However, its low aqueous solubility is the major disadvantage when it comes to in vivo administration. In order to facilitate an aqueous formulation of Nutlin3a and to enhance its apoptotic activity on cancer cells, Nutlin3a was encapsulated in solid lipid nanoparticles (SLNs) prepared by Ouzo method. Physicochemical characterization was performed and activity of apoptosis induction on wild-type P53 expressing LNCaP prostate cancer cell line was evaluated. Nutlin3a-loaded cationic solid lipid nanoparticles were found to stabilize functional P53 at protein level. In addition, induction rate of apoptosis by nanoparticles was higher than Nutlin3a solution in DMSO, proving this nanoparticle formulation is a promising candidate for increasing the efficiency of Nutlin3a for P53(+) cancer cases. Thus, it is anticipated that the results will contribute to evaluate the use of lipid-based nanocarriers to enhance the therapeutic potential of small molecules that are important in cancer cure.

An emerging realization of infectious disease is that pathogens can cause a high incidence of genetic instability within the host as a result of infection-induced DNA lesions. These often lead to classical hallmarks of cancer, one of which is the ability to evade apoptosis despite the presence of numerous genetic mutations that should be otherwise lethal. The Human Immunodeficiency Virus type 1 (HIV-1) is one such pathogen as it induces apoptosis in CD4+ T cells but is largely non-cytopathic in macrophages. As a consequence there is long-term dissemination of the pathogen specifically by these infected yet surviving host cells. Apoptosis is triggered by double-strand breaks (DSBs), such as those induced by integrating retroviruses like HIV-1, and is coordinated by the p53-regulated long noncoding RNA lincRNA-p21. As is typical for a long noncoding RNA, lincRNA-p21 mediates its activities in a complex with one of its two protein binding partners, namely HuR and hnRNP-K. In this work, we monitor the cellular response to infection to determine how HIV-1 induces DSBs in macrophages yet evades apoptosis in these cells. We show that the virus does so by securing the pro-survival MAP2K1/ERK2 cascade early upon entry, in a gp120-dependent manner, to orchestrate a complex dysregulation of lincRNA-p21. By sequestering the lincRNA-p21 partner HuR in the nucleus, HIV-1 enables lincRNA-p21 degradation. Simultaneously, the virus permits transcription of pro-survival genes by sequestering lincRNA-p21\'s other protein partner hnRNP-K in the cytoplasm via the MAP2K1/ERK2 pathway. Of particular note, this MAP2K1/ERK2 pro-survival cascade is switched off during T cell maturation and is thus unavailable for similar viral manipulation in mature CD4+ T cells. We show that the introduction of MAP2K1, ERK2, or HDM2 inhibitors in HIV-infected macrophages results in apoptosis, providing strong evidence that the viral-mediated apoptotic block can be released, specifically by restoring the nuclear interaction of lincRNA-p21 and its apoptosis protein partner hnRNP-K. Together, these results reveal a unique example of pathogenic control over mammalian apoptosis and DNA damage via a host long noncoding RNA, and present MAP2K1/ERK2 inhibitors as a novel therapeutic intervention strategy for HIV-1 infection in macrophages.

The Bcr-Abl tyrosine kinase regulates several Bcl-2 family proteins that confer resistance to apoptosis in chronic myeloid leukemia (CML) cells. Given p53\'s ability to modulate the expression and activity of Bcl-2 family members, we hypothesized that targeting Bcr-Abl, Bcl-2, and p53 concomitantly could have therapeutic benefits in blast crisis (BC) CML and in quiescent CML CD34+ cells that are insensitive to tyrosine kinase inhibitors (TKI). We examined the effects of the MDM2 inhibitor nutlin3a and its combination with the dual Bcl-2 and Bcl-xL inhibitor ABT-737, and the Bcr-Abl inhibitor nilotinib on BC CML patient samples. We found that in quiescent CD34+ progenitors, p53 expression is significantly lower, and MDM2 is higher, compared to their proliferating counterparts. Treatment with nutlin3a induced apoptosis in bulk and CD34+CD38- cells, and in both proliferating and quiescent CD34+ progenitor CML cells. Nutlin3a synergized with ABT-737 and nilotinib, in part by inducing pro-apoptotic, and suppressing anti-apoptotic, Bcl-2 proteins. Nilotinib inhibited the expression of Bcl-xL and Mcl-1 in BC CML cells. These results demonstrate that p53 activation by MDM2 blockade can sensitize BC CML cells, including quiescent CD34+ cells, to Bcl-2 inhibitor- and TKI-induced apoptosis. This novel strategy could be useful in the therapy of BC CML.

For proper development, cells need to coordinate proliferation and cell cycle-exit. This is mediated by a cascade of proteins making sure that each phase of the cell cycle is controlled before the initiation of the next. Retinal progenitor cells divide during the process of interkinetic nuclear migration, where they undergo S-phase on the basal side, followed by mitoses on the apical side of the neuroepithelium. The final cell cycle of chicken retinal horizontal cells (HCs) is an exception to this general cell cycle behavior. Lim1 expressing (+) horizontal progenitor cells (HPCs) have a heterogenic final cell cycle, with some cells undergoing a terminal mitosis on the basal side of the retina. The results in this study show that this terminal basal mitosis of Lim1+ HPCs is not dependent on Chk1/2 for its regulation compared to retinal cells undergoing interkinetic nuclear migration. Neither activating nor blocking Chk1 had an effect on the basal mitosis of Lim1+ HPCs. Furthermore, the Lim1+ HPCs were not sensitive to cisplatin-induced DNA damage and were able to continue into mitosis in the presence of γ-H2AX without activation of caspase-3. However, Nutlin3a-induced expression of p21 did reduce the mitoses, suggesting the presence of a functional p53/p21 response in HPCs. In contrast, the apical mitoses were blocked upon activation of either Chk1/2 or p21, indicating the importance of these proteins during the process of interkinetic nuclear migration. Inhibiting Cdk1 blocked M-phase transition both for apical and basal mitoses. This confirmed that the cyclin B1-Cdk1 complex was active and functional during the basal mitosis of Lim1+ HPCs. The regulation of the final cell cycle of Lim1+ HPCs is of particular interest since it has been shown that the HCs are able to sustain persistent DNA damage, remain in the cell cycle for an extended period of time and, consequently, survive for months.