Angiotensin II-upregulated MAP kinase phosphatase-3 modulates FOXO1 and p21 in adrenocortical H295R cells

MAPK phosphatases (MKP) downregulate the activity of mitogen-activated protein kinases (MAPK), such asERK1/2, and modulate the processes regulated by these kinases. ERK1/2 participate in a wide range of processesincluding tissue-specific hormone-stimulated steroidogenesis. H295R cells are a suitable model for the study ofhuman adrenal cortex functions, particularly steroid synthesis, and respond to angiotensin II (Ang II) triggeringERK1/2 phosphorylation in a transient fashion. MKP-3 dephosphorylates ERK1/2 and, as recently reported,forkhead box protein 1 (FOXO1). Here, we analyzed MKP-3 expression in H295R cells and its putative regulationby Ang II. Results showed the expression of MKP-3 full length (L) and a short splice variant (S), and the upregulationof both isoforms by Ang II. L and S messenger and protein levels increased 30 min after Ang II stimulationand declined over the next 3 h, a temporal frame compatible with ERK1/2 dephosphorylation. In addition, FOXO1activation is known to include its dephosphorylation and nuclear translocation. Therefore, we analyzed the effectof Ang II on FOXO1 modulation. Ang II induced FOXO1 transient phosphorylation and translocation and also theinduction of p21, a FOXO1-dependent gene, whereas MKP-3 knock-down reduced both FOXO1 translocation andp21 induction. These data suggest that, through MKP-3, Ang II counteracts its own effects on ERK1/2 activity andalso triggers the activation of FOXO-1 and the induction of cell cycle inhibitor p21. Taken together, the currentfindings reveal the participation of MKP-3 not only in turn?off but also in turn-on signals which control importantcellular processes.Fil: Mori Sequeiros, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Cohen Sabban, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Dattilo, Melina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Mele, Pablo Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Nudler, Silvana Iris. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Mendez, Carlos Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Maloberti, Paula Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Paz, Cristina del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentin

SI113, a Specific Inhibitor of the Sgk1 Kinase Activity that Counteracts Cancer Cell Proliferation

Background/Aims: Published observations on serum and glucocorticoid regulated kinase 1 (Sgk1) knockout murine models and Sgk1-specific RNA silencing in the RKO human colon carcinoma cell line point to this kinase as a central player in colon carcinogenesis and in resistance to taxanes. Methods: By in vitro kinase activity inhibition assays, cell cycle and viability analysis in human cancer model systems, we describe the biologic effects of a recently identified kinase inhibitor, SI113, characterized by a substituted pyrazolo[3,4-d]pyrimidine scaffold, that shows specificity for Sgk1. Results: SI113 was able to inhibit in vitro cell growth in cancer cells derived from tumors with different origins. In RKO cells, this kinase inhibitor blocked insulin-dependent phosphorylation of the Sgk1 substrate Mdm2, the main regulator of p53 protein stability, and induced necrosis and apoptosis when used as a single agent. Finally, SI113 potentiated the effects of paclitaxel on cell viability. Conclusion: Since SI113 appears to be effective in inducing cell death in RKO cells, potentiating paclitaxel sensitivity, we believe that this new molecule could be efficiently employed, alone or in combination with paclitaxel, in colon cancer chemotherapy

DNA damage and repair biomarkers in cervical cancer patients treated with neoadjuvant chemotherapy: An exploratory analysis

Cervical cancer cells commonly harbour a defective G1/S checkpoint owing to the interaction of viral oncoproteins with p53 and retinoblastoma protein. The activation of the G2/M checkpoint may thus become essential for protecting cancer cells from genotoxic insults, such as chemotherapy. In 52 cervical cancer patients treated with neoadjuvant chemotherapy, we investigated whether the levels of phosphorylated Wee1 (pWee1), a key G2/M checkpoint kinase, and y-H2AX, a marker of DNA double-strand breaks, discriminated between patients with a pathological complete response (pCR) and those with residual disease. We also tested the association between pWee1 and phosphorylated Chk1 (pChk1), a kinase acting upstream Wee1 in the G2/M checkpoint pathway. pWee1, y-H2AX and pChk1 were retrospectively assessed in diagnostic biopsies by immunohistochemistry. The degrees of pWee1 and pChk1 expression were defined using three different classification methods, i.e., staining intensity, Allred score, and a multiplicative score. y-H2AX was analyzed both as continuous and categorical variable. Irrespective of the classification used, elevated levels of pWee1 and y-H2AX were significantly associated with a lower rate of pCR. In univariate and multivariate analyses, pWee1 and y-H2AX were both associated with reduced pCR. Internal validation conducted through a re-sampling without replacement procedure confirmed the robustness of the multivariate model. Finally, we found a significant association between pWee1 and pChk1. The message conveyed by the present analysis is that biomarkers of DNA damage and repair may predict the efficacy of neoadjuvant chemotherapy in cervical cancer. Further studies are warranted to prospectively validate these encouraging findings

SGK1, the New Player in the Game of Resistance: Chemo-Radio Molecular Target and Strategy for Inhibition

The serum- and glucocorticoid-regulated kinase (SGK) family consists of three members, SGK1, SGK2 and SGK3, all displaying serine/threonine kinase activity and sharing structural and functional similarities with the AKT family of kinases. SGK1 was originally described as a key enzyme in the hormonal regulation of several ion channels and pumps. Over time, growing and impressive evidence has been accumulated, linking SGK1 to the cell survival, de-differentiation, cell cycle control, regulation of caspases, response to chemical, mechanical and oxidative injury in cancer models as well as to the control of mitotic stability. Much evidence shows that SGK1 is over-expressed in a variety of epithelial tumors. More recently, many contributions to the published literature demonstrate that SGK1 can mediate chemo-and radio-resistance during the treatment of various human tumors, both in vitro and in vivo. SGK1 appears therefore as a dirty player in the stress response to chemical and radio-agents, responsible of a selective advantage that favors the uncontrolled tumor progression and the selection of the most aggressive clones. The purpose of this review is the analysis of the literature describing SGK1 as central node of the cell resistance, and a summary of the possible strategies in the pharmacological targeting of SGK1

Stimulation of Purinergic Receptors Modulates Chemokine Expression in Human Keratinocytes

ATP is abundantly released from stressed or damaged cells in response to mechanical stimulation, bacteria, or noxious agents. In this study, we have investigated the possible involvement of P2 receptors (receptor for extracellular nucleotides) in the expression and release of inflammatory mediators by human keratinocytes. Notably, extracellular ATP displayed a complex regulation of IFN-γ-stimulated chemokine expression, with upregulation of CC chemokine ligand 2 (CCL2), CCL5 and CXC chemokine ligand 8 (CXCL8), and suppression of the receptor CXC chemokine receptor 3 (CXCR3), CXCL9, CXCL10, and CXCL11. The effect of ATP was mimicked by ADP and adenosine-5′-O-3-thiotriphosphate, whereas 2′,3′-O-(4-benzoylbenzoyl) ATP (BzATP) downmodulated all chemokines investigated. UTP had no effect on IFN-γ-stimulated chemokine secretion. The broad-spectrum P2 receptor antagonist suramin and the selective P2Y1 inhibitor adenosine 3′-phosphate 5′-phosphosulfate counteracted the effect of ATP on secretion of all the chemokines examined, whereas pyridoxal phosphate 6-azophenyl 2′,4′-disulfonic acid and KN62 (1-[N,O-bis(5-isoquinoline sulfonyl)-N-methyl-L-tyrosyl] 4 phenylpiperazine) partially prevented the inhibitory effect of ATP on CXCL10 secretion, but on the other hand potentiated the ATP-stimulatory effect on CCL5, CCL2, and CXCL8 release. In lesional skin of psoriasis and atopic dermatitis patients, intense P2X7 reactivity was confined to the cell membrane of the basal layer, whereas diffuse P2Y1 immunostaining was found throughout the epidermis. Collectively, our data suggest that the orchestrated activation of distinct P2Y and P2X receptors modulates skin inflammation

A Novel Splicing Variant of COL2A1 in a Fetus with Achondrogenesis Type II: Interpretation of Pathogenicity of In-Frame Deletions

Achondrogenesis type II (ACG2) is a lethal skeletal dysplasia caused by dominant pathogenic variants in COL2A1. Most of the variants found in patients with ACG2 affect the glycine residue included in the Gly-X-Y tripeptide repeat that characterizes the type II collagen helix. In this study, we reported a case of a novel splicing variant of COL2A1 in a fetus with ACG2. An NGS analysis of fetal DNA revealed a heterozygous variant c.1267-2_1269del located in intron 20/exon 21. The variant occurred de novo since it was not detected in DNA from the blood samples of parents. We generated an appropriate minigene construct to study the effect of the variant detected. The minigene expression resulted in the synthesis of a COL2A1 messenger RNA lacking exon 21, which generated a predicted in-frame deleted protein. Usually, in-frame deletion variants of COL2A1 cause a phenotype such as Kniest dysplasia, which is milder than ACG2. Therefore, we propose that the size and position of an in-frame deletion in COL2A1 may be relevant in determining the phenotype of skeletal dysplasia

SARS-CoV-2 Infection Alters the Phenotype and Gene Expression of Adipocytes

Epidemiological evidence emphasizes that excess fat mass is associated with an increased risk of severe COVID-19 disease. Nevertheless, the intricate interplay between SARS-CoV-2 and adipocytes remains poorly understood. It is crucial to decipher the progression of COVID-19 both in the acute phase and on long-term outcomes. In this study, an in vitro model using the human SGBS cell line (Simpson-Golabi-Behmel syndrome) was developed to investigate the infectivity of SARS-CoV-2 in adipocytes, and the effects of virus exposure on adipocyte function. Our results show that SGBS adipocytes expressing ACE2 are susceptible to SARS-CoV-2 infection, as evidenced by the release of the viral genome into the medium, detection of the nucleocapsid in cell lysates, and positive immunostaining for the spike protein. Infected adipocytes show remarkable changes compared to uninfected controls: increased surface area of lipid droplets, upregulated expression of genes of inflammation (Haptoglobin, MCP-1, IL-6, PAI-1), increased oxidative stress (MnSOD), and a concomitant reduction of transcripts related to adipocyte function (leptin, fatty acid synthase, perilipin). Moreover, exogenous expression of spike protein in SGBS adipocytes also led to an increase in lipid droplet size. In conclusion using the human SGBS cell line, we detected SARS-CoV-2 infectivity in adipocytes, revealing substantial morphological and functional changes in infected cells