Subscaling of a cytosolic RNA binding protein governs cell size homeostasis in the multiple fission alga Chlamydomonas.

Coordination of growth and division in eukaryotic cells is essential for populations of proliferating cells to maintain size homeostasis, but the underlying mechanisms that govern cell size have only been investigated in a few taxa. The green alga Chlamydomonas reinhardtii (Chlamydomonas) proliferates using a multiple fission cell cycle that involves a long G1 phase followed by a rapid series of successive S and M phases (S/M) that produces 2n daughter cells. Two control points show cell-size dependence: the Commitment control point in mid-G1 phase requires the attainment of a minimum size to enable at least one mitotic division during S/M, and the S/M control point where mother cell size governs cell division number (n), ensuring that daughter distributions are uniform. tny1 mutants pass Commitment at a smaller size than wild type and undergo extra divisions during S/M phase to produce small daughters, indicating that TNY1 functions to inhibit size-dependent cell cycle progression. TNY1 encodes a cytosolic hnRNP A-related RNA binding protein and is produced once per cell cycle during S/M phase where it is apportioned to daughter cells, and then remains at constant absolute abundance as cells grow, a property known as subscaling. Altering the dosage of TNY1 in heterozygous diploids or through mis-expression increased Commitment cell size and daughter cell size, indicating that TNY1 is a limiting factor for both size control points. Epistasis placed TNY1 function upstream of the retinoblastoma tumor suppressor complex (RBC) and one of its regulators, Cyclin-Dependent Kinase G1 (CDKG1). Moreover, CDKG1 protein and mRNA were found to over-accumulate in tny1 cells suggesting that CDKG1 may be a direct target of repression by TNY1. Our data expand the potential roles of subscaling proteins outside the nucleus and imply a control mechanism that ties TNY1 accumulation to pre-division mother cell size.

Specific cellular microenvironments for spatiotemporal regulation of StAR and steroid synthesis.

For many years, research in the field of steroid synthesis has aimed to understand the regulation of the rate-limiting step of steroid synthesis, i.e. the transport of cholesterol from the outer to the inner mitochondrial membrane, and identify the protein involved in the conversion of cholesterol into pregnenolone. The extraordinary work by B Clark, J Wells, S R King, and D M Stocco eventually identified this protein and named it steroidogenic acute regulatory protein (StAR). The group's finding was also one of the milestones in understanding the mechanism of nonvesicular lipid transport between organelles. A notable feature of StAR is its high degree of phosphorylation. In fact, StAR phosphorylation in the acute phase is required for full steroid biosynthesis. As a contribution to this subject, our work has led to the characterization of StAR as a substrate of kinases and phosphatases and as an integral part of a mitochondrion-associated multiprotein complex, essential for StAR function and cholesterol binding and mitochondrial transport to yield maximum steroid production. Results allow us to postulate the existence of a specific cellular microenvironment where StAR protein synthesis and activation, along with steroid synthesis and secretion, are performed in a compartmentalized manner, at the site of hormone receptor stimulation, and involving the compartmentalized formation of the steroid molecule-synthesizing complex.

Functionality of bone marrow mesenchymal stromal cells derived from head and neck cancer patients - A FDA-IND enabling study regarding MSC-based treatments for radiation-induced xerostomia.

PURPOSE: Salivary dysfunction is a significant side effect of radiation therapy for head and neck cancer (HNC). Preliminary data suggests that mesenchymal stromal cells (MSCs) can improve salivary function. Whether MSCs from HNC patients who have completed chemoradiation are functionally similar to those from healthy patients is unknown. We performed a pilot clinical study to determine whether bone marrow-derived MSCs [MSC(M)] from HNC patients could be used for the treatment of RT-induced salivary dysfunction. METHODS: An IRB-approved pilot clinical study was undertaken on HNC patients with xerostomia who had completed treatment two or more years prior. Patients underwent iliac crest bone marrow aspirate and MSC(M) were isolated and cultured. Culture-expanded MSC(M) were stimulated with IFNγ and cryopreserved prior to reanimation and profiling for functional markers by flow cytometry and ELISA. MSC(M) were additionally injected into mice with radiation-induced xerostomia and the changes in salivary gland histology and salivary production were examined. RESULTS: A total of six subjects were enrolled. MSC(M) from all subjects were culture expanded to > 20 million cells in a median of 15.5 days (range 8-20 days). Flow cytometry confirmed that cultured cells from HNC patients were MSC(M). Functional flow cytometry demonstrated that these IFNγ-stimulated MSC(M) acquired an immunosuppressive phenotype. IFNγ-stimulated MSC(M) from HNC patients were found to express GDNF, WNT1, and R-spondin 1 as well as pro-angiogenesis and immunomodulatory cytokines. In mice, IFNγ-stimulated MSC(M) injection after radiation decreased the loss of acinar cells, decreased the formation of fibrosis, and increased salivary production. CONCLUSIONS: MSC (M) from previously treated HNC patients can be expanded for auto-transplantation and are functionally active. Furthermore IFNγ-stimulated MSC(M) express proteins implicated in salivary gland regeneration. This study provides preliminary data supporting the feasibility of using autologous MSC(M) from HNC patients to treat RT-induced salivary dysfunction.

Supervisión de salud de niños/as con Síndrome de Down realizado por enfermera, desde la mirada de sus cuidadores / Caregivers' perspectives about the health supervision of children with Down Syndrome carried out by a nurse

INTRODUCCION: Los niños con Síndrome de Down (SD) y sus cuidadores requieren de una atención de salud multidisciplinaria que responda a sus necesidades desde una perspectiva integral. OBJETIVO: conocer las experiencias y expectativas que tienen los cuidadores de niños con SD en relación con los cuidados brindados por la enfermera durante el control de supervisión de salud infantil. METOLOGÍA: Estudio cualitativo fenomenológico, muestra intencionada de casos por criterios conformada por 11 cuidadores de niños con SD entre 0 y 9 años, que recibieron prestaciones en el Control de Salud Infantil. Se realizaron entrevistas semiestructuradas, los datos se analizaron siguiendo el esquema de reducción progresiva. Contó con la aprobación de la Dirección de Carrera de Enfermería, Universidad Santo Tomás (Temuco-Chile) (Acta 07-2018). RESULTADOS: se generaron dos categorías: "Experiencia de atención otorgada por la enfermera" y "Expectativas vinculadas a la atención proporcionada por la enfermera", se develó que los cuidadores de niños con SD perciben que los cuidados brindados por la enfermera durante la atención son insuficientes en relación con las necesidades que estos niños presentan. Esto estaría vinculado con la falta de conocimientos sobre esta condición y su abordaje integral. Con respecto a sus expectativas, refieren necesidad de acompañamiento, apoyo, educación y de profesionales de enfermería capacitados. CONCLUSIONES: urge abordar las brechas con respecto a las competencias del profesional de enfermería que realiza controles de salud del niño para poder entregar una atención de calidad a ellos y sus familias.

INTRODUCTION: Children with Down Syndrome (DS) and their caregivers require multidisciplinary health care that meets their needs from a comprehensive perspective. OBJECTIVE: to know the experiences and expectations that caregivers of children with DS have concerning the care provided by the nurse in the Child Health Supervision Attention. METHOD: A qualitative phenomenological study was conducted, an intentional sample of cases by criteria made up of 11 caregivers of children with DS between 0 and 9 years of age, who received benefits at the Child Health Control. Semi-structured interviews were carried out; the data were analyzed following the progressive reduction scheme. It had the approval of the Nursing Career Department, Santo Tomás University (Temuco-Chile) (Acta 07-2018). RESULTS: two categories were generated: "Experience of care provided by the nurse" and "Expectations linked to the care provided by the nurse", it was revealed that caregivers of children with DS perceive that the care provided by the nurse during the control is insufficient with the children's needs also, data showed a link to the lack of knowledge about this condition and its comprehensive approach. Regarding their expectations, they report the need for accompaniment, support, and education from the nurse, in addition to being a trained professional. CONCLUSION: It is urgent to address the gaps regarding the competencies of the nursing professional who performs child health checks to deliver quality care to them and their families.

Single cell profiling at the maternal-fetal interface reveals a deficiency of PD-L1+ non-immune cells in human spontaneous preterm labor.

The mechanisms that underlie the timing of labor in humans are largely unknown. In most pregnancies, labor is initiated at term (≥ 37 weeks gestation), but in a signifiicant number of women spontaneous labor occurs preterm and is associated with increased perinatal mortality and morbidity. The objective of this study was to characterize the cells at the maternal-fetal interface (MFI) in term and preterm pregnancies in both the laboring and non-laboring state in Black women, who have among the highest preterm birth rates in the U.S. Using mass cytometry to obtain high-dimensional single-cell resolution, we identified 31 cell populations at the MFI, including 25 immune cell types and six non-immune cell types. Among the immune cells, maternal PD1+ CD8 T cell subsets were less abundant in term laboring compared to term non-laboring women. Among the non-immune cells, PD-L1+ maternal (stromal) and fetal (extravillous trophoblast) cells were less abundant in preterm laboring compared to term laboring women. Consistent with these observations, the expression of CD274, the gene encoding PD-L1, was significantly depressed and less responsive to fetal signaling molecules in cultured mesenchymal stromal cells from the decidua of preterm compared to term women. Overall, these results suggest that the PD1/PD-L1 pathway at the MFI may perturb the delicate balance between immune tolerance and rejection and contribute to the onset of spontaneous preterm labor.

New insights into signal transduction pathways in adrenal steroidogenesis: role of mitochondrial fusion, lipid mediators, and MAPK phosphatases.

Hormone-receptor signal transduction has been extensively studied in adrenal gland. Zona glomerulosa and fasciculata cells are responsible for glucocorticoid and mineralocorticoid synthesis by adrenocorticotropin (ACTH) and angiotensin II (Ang II) stimulation, respectively. Since the rate-limiting step in steroidogenesis occurs in the mitochondria, these organelles are key players in the process. The maintenance of functional mitochondria depends on mitochondrial dynamics, which involves at least two opposite events, i.e., mitochondrial fusion and fission. This review presents state-of-the-art data on the role of mitochondrial fusion proteins, such as mitofusin 2 (Mfn2) and optic atrophy 1 (OPA1), in Ang II-stimulated steroidogenesis in adrenocortical cells. Both proteins are upregulated by Ang II, and Mfn2 is strictly necessary for adrenal steroid synthesis. The signaling cascades of steroidogenic hormones involve an increase in several lipidic metabolites such as arachidonic acid (AA). In turn, AA metabolization renders several eicosanoids released to the extracellular medium able to bind membrane receptors. This report discusses OXER1, an oxoeicosanoid receptor which has recently arisen as a novel participant in adrenocortical hormone-stimulated steroidogenesis through its activation by AA-derived 5-oxo-ETE. This work also intends to broaden knowledge of phospho/dephosphorylation relevance in adrenocortical cells, particularly MAP kinase phosphatases (MKPs) role in steroidogenesis. At least three MKPs participate in steroid production and processes such as the cellular cycle, either directly or by means of MAP kinase regulation. To sum up, this review discusses the emerging role of mitochondrial fusion proteins, OXER1 and MKPs in the regulation of steroid synthesis in adrenal cortex cells.

Quantification of very late xerostomia in head and neck cancer patients after irradiation.

Objective: Radiation therapy (RT) for head and neck cancer (HNC) can result in severe xerostomia, or the subjective feeling of dry mouth. Characterizing xerostomia is critical to designing future clinical trials investigating how to improve HNC patients' quality of life (QoL). Few studies have investigated the very late (>5 years post-RT) effects of RT for HNC. We undertook preliminary studies quantifying very late xerostomia. Methods: Six adults who underwent RT for HNC at least 5 years prior and reported xerostomia were enrolled. Five healthy adults without a self-reported history of HNC or xerostomia were enrolled as controls. All participants completed three validated surveys to measure xerostomia-related QoL. Salivary production rates were measured and compositional analysis of the saliva and oral microbiome was completed. Results: The QoL survey scores for the HNC participants were significantly worse as compared to the control participants. The HNC participants produced less unstimulated saliva (p = .02) but not less stimulated saliva. The median salivary mucin significantly higher in HNC participants than in control participants (p = .02). There was no significant difference between the pH, amylase, or total protein. Microbiome analysis revealed alpha diversity to be significantly lower in the HNC participants. Conclusion: In the survivors of HNC who suffer from late toxicities, multiple means of measuring toxicity may be useful. We found that in patients with radiation-induced xerostomia over 5 years after therapy, not only were the QoL surveys significantly worse, as expected, but other measurements such as mucin and oral microbiome diversity were also significantly different. Level of evidence: 3.

Microencapsulated Isoniazid-Loaded Metal-Organic Frameworks for Pulmonary Administration of Antituberculosis Drugs.

Tuberculosis (TB) is an infectious disease that causes a great number of deaths in the world (1.5 million people per year). This disease is currently treated by administering high doses of various oral anti-TB drugs for prolonged periods (up to 2 years). While this regimen is normally effective when taken as prescribed, many people with TB experience difficulties in complying with their medication schedule. Furthermore, the oral administration of standard anti-TB drugs causes severe side effects and widespread resistances. Recently, we proposed an original platform for pulmonary TB treatment consisting of mannitol microspheres (Ma MS) containing iron (III) trimesate metal-organic framework (MOF) MIL-100 nanoparticles (NPs). In the present work, we loaded this system with the first-line anti-TB drug isoniazid (INH) and evaluated both the viability and safety of the drug vehicle components, as well as the cell internalization of the formulation in alveolar A549 cells. Results show that INH-loaded MOF (INH@MIL-100) NPs were efficiently microencapsulated in Ma MS, which displayed suitable aerodynamic characteristics for pulmonary administration and non-toxicity. MIL-100 and INH@MIL-100 NPs were efficiently internalized by A549 cells, mainly localized in the cytoplasm. In conclusion, the proposed micro-nanosystem is a good candidate for the pulmonary administration of anti-TB drugs.

Características clínicas, epidemiológicas y patológicas de los linfomas en pacientes del Instituto Nacional de Salud del Niño de Breña - Perú, 2015-2019

Objetivo. Determinar las características clínicas, epidemiológicas y patológicas de los linfomas en pacientes del Instituto Nacional de Salud del Niño Breña entre el 2015-2019. Métodos. Se realizó un estudio cuantitativo, observacional, transversal, descriptivo y retrospectivo; correspondiente a los pacientes pediátricos con diagnóstico de linfoma entre los años 2015 al 2019. Para el análisis se utilizó el programa SPSS v24. Las variables cuantitativas fueron expresadas en medidas de tendencia central y las cualitativas en frecuencias absolutas y relativas. Resultados. Se incluyeron 43 casos, el promedio fue 7,2 años (1a - 16a), el 72,1% (n= 31) fueron del sexo masculino. Entre los síntomas más frecuentes están fiebre 51,2% (n= 22), baja de peso 41,9% (n= 18) y cansancio 20,9% (n= 9) y entre los signos prevalentes, las linfadenopatías 83,7% (n=36) y tumoración abdominal con 14% (n= 6). La localización más frecuente fue la de tipo ganglionar con 69,8% (n=30). La anemia se presentó en un 58,1% (n= 25). Según el diagnóstico anátomopatologico, 20 (48,8%) casos fueron Linfoma Hodgkin (LH) y 23 (51,2%) no Hodgkin (LNH); siendo los tipos más frecuente el linfoma linfoblástico B con un 25,6% (n=11), seguido de LH celularidad mixta con 20,9% (n= 9). El linfoma más frecuente en etapa preescolar fue el LH celularidad mixta con 31,3% (n= 5), mientras que en etapa escolar el linfoma linfoblástico B con 25,9% (n=7). Conclusiones. En pacientes pediátricos, el linfoma afectó mayormente al sexo masculino, presentando fiebre, baja de peso, linfoadenopatías, tumoración abdominal y anemia. El linfoma linfoblástico B fue el más frecuente; estas características clínicas son similares a lo reportado a nivel mundial.

Objective. To determine the clinical, epidemiological and pathological characteristics of lymphomas in patients of the National Institute of Child Health Breña between 2015-2019. Methods. A quantitative, observational, crosssectional, descriptive and retrospective study was carried out; corresponding to pediatric patients diagnosed with lymphoma between 2015 and 2019. The SPSS v24 program was used for the analysis. The quantitative variables were expressed in measures of central tendency and the qualitative ones in absolute and relative frequencies. Results. 43 cases were included, the average age was 7.2 years (1a - 16a), 72.1% (n = 31) were male. Among the most frequent symptoms are fever 51.2% (n = 22), weight loss 41.9% (n = 18) and fatigue 20.9% (n = 9) and among the prevalent signs, lymphadenopathy 83.7% (n = 36) and abdominal mass with 14% (n = 6). The most frequent location was the lymph node type with 69.8% (n = 30). Anemia occurred in 58.1% (n = 25). According to the pathological diagnosis, 20 (48.8%) cases were Hodgkin's Lymphoma (HL) and 23 (51.2%) were non-Hodgkin's (NHL); the most frequent types being B lymphoblastic lymphoma with 25.6% (n = 11), followed by mixed cellularity LH with 20.9% (n = 9). The most frequent lymphoma in preschool stage was mixed cellularity LH with 31.3% (n = 5), while in school stage B lymphoblastic lymphoma with 25.9% (n = 7). Conclusions. In pediatric patients, lymphoma mainly affects the male sex, presenting fever, weight loss, lymphadenopathy, abdominal tumor and anemia. B lymphoblastic lymphoma was the most frequent; these clinical characteristics are similar to those reported worldwide.

Regulation and role of Acyl-CoA synthetase 4 in glial cells.

Acyl-CoA synthetase 4 (Acsl4), an enzyme involved in arachidonic acid (AA) metabolism, participates in physiological and pathological processes such as steroidogenesis and cancer. The role of Acsl4 in neurons and in nervous system development has also been documented but little is known regarding its functionality in glial cells. In turn, several processes in glial cells, including neurosteroidogenesis, stellation and AA uptake, are regulated by cyclic adenosine monophosphate (cAMP) signal. In this context, the aim of this work was to analyze the expression and functional role of Acsl4 in primary rat astrocyte cultures and in the C6 glioma cell line by chemical inhibition and stable silencing, respectively. Results show that Acsl4 expression was regulated by cAMP in both models and that cAMP stimulation of steroidogenic acute regulatory protein mRNA levels was reduced by Acsl4 inhibition or silencing. Also, Acsl4 inhibition reduced progesterone synthesis stimulated by cAMP and also affected cAMP-induced astrocyte stellation, decreasing process elongation and increasing branching complexity. Similar effects were observed for Acsl4 silencing on cAMP-induced C6 cell morphological shift. Moreover, Acsl4 inhibition and silencing reduced proliferation and migration of both cell types. Acsl4 silencing in C6 cells reduced the capacity for colony proliferation and neurosphere formation, the latter ability also being abolished by Acsl4 inhibition. In sum, this work presents novel evidence of Acsl4 involvement in neurosteroidogenesis and the morphological changes of glial cells promoted by cAMP. Furthermore, Acsl4 participates in migration and proliferation, also affecting cell self-renewal. Altogether, these findings provide insights into Acsl4 functions in glial cells.

Transcriptome and regulatory maps of decidua-derived stromal cells inform gene discovery in preterm birth.

While a genetic component of preterm birth (PTB) has long been recognized and recently mapped by genome-wide association studies (GWASs), the molecular determinants underlying PTB remain elusive. This stems in part from an incomplete availability of functional genomic annotations in human cell types relevant to pregnancy and PTB. We generated transcriptome (RNA-seq), epigenome (ChIP-seq of H3K27ac, H3K4me1, and H3K4me3 histone modifications), open chromatin (ATAC-seq), and chromatin interaction (promoter capture Hi-C) annotations of cultured primary decidua-derived mesenchymal stromal/stem cells and in vitro differentiated decidual stromal cells and developed a computational framework to integrate these functional annotations with results from a GWAS of gestational duration in 56,384 women. Using these resources, we uncovered additional loci associated with gestational duration and target genes of associated loci. Our strategy illustrates how functional annotations in pregnancy-relevant cell types aid in the experimental follow-up of GWAS for PTB and, likely, other pregnancy-related conditions.

Metal-Organic Framework Microsphere Formulation for Pulmonary Administration.

Although nanoscaled metal-organic frameworks (nanoMOFs) are promising drug carriers, their appropriate formulation remains almost unexplored and basically restricted to intravenous routes. Lungs, beneficiating from a large absorption surface and low enzymatic presence, are a very attractive target for both local and systemic delivery. However, pulmonary nanoMOF formulation is a pending and defying task. Thus, we propose a pioneer nanoMOF-based microsphere system as a potential platform for pulmonary administration. A biocompatible nanoMOF was successfully encapsulated in mannitol by a simple and continuous spray-drying technique. Upon intratracheal administration to rats, the resulting formulation, exhibiting optimal properties (i.e., homogeneity, size, density, and spray-drying process yield), was able to release the intact nanoMOF carrier uniformly along the lungs, reaching the bronchioles and alveoli.

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 as ERK1/2, and modulate the processes regulated by these kinases. ERK1/2 participate in a wide range of processes including tissue-specific hormone-stimulated steroidogenesis. H295R cells are a suitable model for the study of human adrenal cortex functions, particularly steroid synthesis, and respond to angiotensin II (Ang II) triggering ERK1/2 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 regulation by Ang II. Results showed the expression of MKP-3 full length (L) and a short splice variant (S), and the upregulation of both isoforms by Ang II. L and S messenger and protein levels increased 30 min after Ang II stimulation and declined over the next 3 h, a temporal frame compatible with ERK1/2 dephosphorylation. In addition, FOXO1 activation is known to include its dephosphorylation and nuclear translocation. Therefore, we analyzed the effect of Ang II on FOXO1 modulation. Ang II induced FOXO1 transient phosphorylation and translocation and also the induction of p21, a FOXO1-dependent gene, whereas MKP-3 knock-down reduced both FOXO1 translocation and p21 induction. These data suggest that, through MKP-3, Ang II counteracts its own effects on ERK1/2 activity and also triggers the activation of FOXO-1 and the induction of cell cycle inhibitor p21. Taken together, the current findings reveal the participation of MKP-3 not only in turn-off but also in turn-on signals which control important cellular processes.

Patient Derived Models to Study Head and Neck Cancer Radiation Response.

Patient-derived model systems are important tools for studying novel anti-cancer therapies. Patient-derived xenografts (PDXs) have gained favor over the last 10 years as newer mouse strains have improved the success rate of establishing PDXs from patient biopsies. PDXs can be engrafted from head and neck cancer (HNC) samples across a wide range of cancer stages, retain the genetic features of their human source, and can be treated with both chemotherapy and radiation, allowing for clinically relevant studies. Not only do PDXs allow for the study of patient tissues in an in vivo model, they can also provide a renewable source of cancer cells for organoid cultures. Herein, we review the uses of HNC patient-derived models for radiation research, including approaches to establishing both orthotopic and heterotopic PDXs, approaches and potential pitfalls to delivering chemotherapy and radiation to these animal models, biological advantages and limitations, and alternatives to animal studies that still use patient-derived tissues.

Identification of Chlamydomonas reinhardtii endogenous genic flanking sequences for improved transgene expression.

Chlamydomonas reinhardtii is a unicellular green alga that has attracted interest due to its potential biotechnological applications, and as a model for algal biofuel and energy metabolism. Despite all the advantages that this unicellular alga offers, poor and inconsistent expression of nuclear transgenes remains an obstacle for basic and applied research. We used a data-mining strategy to identify highly expressed genes in Chlamydomonas whose flanking sequences were tested for the ability to drive heterologous nuclear transgene expression. Candidates identified in this search included two ribosomal protein genes, RPL35a and RPL23, and ferredoxin, FDX1, whose flanking regions including promoters, terminators and untranslated sequences could drive stable luciferase transgene expression to significantly higher levels than the commonly used Hsp70A-RBCS2 (AR) hybrid promoter/terminator sequences. The RPL23 flanking sequences were further tested using the zeocin resistance gene sh-ble as a reporter in monocistronic and dicistronic constructs, and consistently yielded higher numbers of zeocin-resistant transformants and higher levels of resistance than AR- or PSAD-based vectors. Chlamydomonas RPL23 sequences also enabled transgene expression in Volvox carteri. Our study provides an additional benchmark for strong constitutive expression of transgenes in Chlamydomonas, and develops a general approach for identifying flanking sequences that can be used to drive transgene expression for any organism where transcriptome data are available.

Role of Protein Phosphorylation and Tyrosine Phosphatases in the Adrenal Regulation of Steroid Synthesis and Mitochondrial Function.

In adrenocortical cells, adrenocorticotropin (ACTH) promotes the activation of several protein kinases. The action of these kinases is linked to steroid production, mainly through steroidogenic acute regulatory protein (StAR), whose expression and activity are dependent on protein phosphorylation events at genomic and non-genomic levels. Hormone-dependent mitochondrial dynamics and cell proliferation are functions also associated with protein kinases. On the other hand, protein tyrosine dephosphorylation is an additional component of the ACTH signaling pathway, which involves the "classical" protein tyrosine phosphatases (PTPs), such as Src homology domain (SH) 2-containing PTP (SHP2c), and members of the MAP kinase phosphatase (MKP) family, such as MKP-1. PTPs are rapidly activated by posttranslational mechanisms and participate in hormone-stimulated steroid production. In this process, the SHP2 tyrosine phosphatase plays a crucial role in a mechanism that includes an acyl-CoA synthetase-4 (Acsl4), arachidonic acid (AA) release and StAR induction. In contrast, MKPs in steroidogenic cells have a role in the turn-off of the hormonal signal in ERK-dependent processes such as steroid synthesis and, perhaps, cell proliferation. This review analyzes the participation of these tyrosine phosphates in the ACTH signaling pathway and the action of kinases and phosphatases in the regulation of mitochondrial dynamics and steroid production. In addition, the participation of kinases and phosphatases in the signal cascade triggered by different stimuli in other steroidogenic tissues is also compared to adrenocortical cell/ACTH and discussed.

ACTH Modulates PTP-PEST Activity and Promotes Its Interaction With Paxillin.

Adrenocorticotropic hormone (ACTH) treatment has been proven to promote paxillin dephosphorylation and increase soluble protein tyrosine phosphatase (PTP) activity in rat adrenal zona fasciculata (ZF). Also, in-gel PTP assays have shown the activation of a 115-kDa PTP (PTP115) by ACTH. In this context, the current work presents evidence that PTP115 is PTP-PEST, a PTP that recognizes paxillin as substrate. PTP115 was partially purified from rat adrenal ZF and PTP-PEST was detected through Western blot in bioactive samples taken in each purification step. Immunohistochemical and RT-PCR studies revealed PTP-PEST expression in rat ZF and Y1 adrenocortical cells. Moreover, a PTP-PEST siRNA decreased the expression of this phosphatase. PKA phosphorylation of purified PTP115 isolated from non-ACTH-treated rats increased KM and VM . Finally, in-gel PTP assays of immunoprecipitated paxillin from control and ACTH-treated rats suggested a hormone-mediated increase in paxillin-PTP115 interaction, while PTP-PEST and paxillin co-localize in Y1 cells. Taken together, these data demonstrate PTP-PEST expression in adrenal ZF and its regulation by ACTH/PKA and also suggest an ACTH-induced PTP-PEST-paxillin interaction. J. Cell. Biochem. 117: 2170-2181, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division.

Proliferating cells actively control their size by mechanisms that are poorly understood. The unicellular green alga Chlamydomonas reinhardtii divides by multiple fission, wherein a 'counting' mechanism couples mother cell-size to cell division number allowing production of uniform-sized daughters. We identified a sizer protein, CDKG1, that acts through the retinoblastoma (RB) tumor suppressor pathway as a D-cyclin-dependent RB kinase to regulate mitotic counting. Loss of CDKG1 leads to fewer mitotic divisions and large daughters, while mis-expression of CDKG1 causes supernumerous mitotic divisions and small daughters. The concentration of nuclear-localized CDKG1 in pre-mitotic cells is set by mother cell size, and its progressive dilution and degradation with each round of cell division may provide a link between mother cell-size and mitotic division number. Cell-size-dependent accumulation of limiting cell cycle regulators such as CDKG1 is a potentially general mechanism for size control.

cAMP-activated Nr4a1 expression requires ERK activity and is modulated by MAPK phosphatase-1 in MA-10 Leydig cells.

In Leydig cells, LH and cAMP promote ERK1/2 activation and MAPK phosphatase-1 (MKP-1) induction. MKP-1 up-regulation, which involves post-translational modifications such as ERK1/2-mediated phosphorylation, reduces ERK1/2 phosphorylation as well as Steroidogenic Acute Regulatory (StAR) protein expression and steroidogenesis. As LH- and cAMP-promoted StAR transcription requires the induction of Nur77, product of Nr4a1 gene, we analyzed the roles of ERK1/2 and MKP-1 in 8Br-cAMP-mediated Nr4a1 expression in MA-10 Leydig cells. Pharmacological blockade of ERK1/2 activation partially reduced the 8Br-cAMP-mediated increase in both Nr4a1 messenger levels and promoter activity. MKP-1 knock-down increased 8Br-cAMP-induced promoter activity, while its over-expression produced the opposite effect. It is concluded that Nr4a1 induction is dependent on ERK1/2 and that MKP-1 negatively regulates this induction. Experiments based on the over-expression of MKP-1 mutated forms revealed that MKP-1 half life is determined by post-translational modifications in ERK-consensus sites, a regulation that modulates the effect of MKP-1 on Nr4a1 expression.

Defects in a new class of sulfate/anion transporter link sulfur acclimation responses to intracellular glutathione levels and cell cycle control.

We previously identified a mutation, suppressor of mating type locus3 15-1 (smt15-1), that partially suppresses the cell cycle defects caused by loss of the retinoblastoma tumor suppressor-related protein encoded by the MAT3 gene in Chlamydomonas reinhardtii. smt15-1 single mutants were also found to have a cell cycle defect leading to a small-cell phenotype. SMT15 belongs to a previously uncharacterized subfamily of putative membrane-localized sulfate/anion transporters that contain a sulfate transporter domain and are found in a widely distributed subset of eukaryotes and bacteria. Although we observed that smt15-1 has a defect in acclimation to sulfur-limited growth conditions, sulfur acclimation (sac) mutants, which are more severely defective for acclimation to sulfur limitation, do not have cell cycle defects and cannot suppress mat3. Moreover, we found that smt15-1, but not sac mutants, overaccumulates glutathione. In wild-type cells, glutathione fluctuated during the cell cycle, with highest levels in mid G1 phase and lower levels during S and M phases, while in smt15-1, glutathione levels remained elevated during S and M. In addition to increased total glutathione levels, smt15-1 cells had an increased reduced-to-oxidized glutathione redox ratio throughout the cell cycle. These data suggest a role for SMT15 in maintaining glutathione homeostasis that impacts the cell cycle and sulfur acclimation responses.