bims-bicyki 2021-10-17 papers

bims-bicyki

Biomed News

on Bicaudal-C1 and interactors in cystic kidney disease

Issue of 2021‒10‒17
twenty papers selected by
Céline Gagnieux
École Polytechnique Fédérale de Lausanne (EPFL)

Celiac artery dissection in polycystic kidney disease.
Renal plasticity revealed through reversal of polycystic kidney disease in mice.
Role of DNA-Dependent Protein Kinase in Mediating Cyst Growth in Autosomal Dominant Polycystic Kidney Disease.
The tyrosine-kinase inhibitor Nintedanib ameliorates autosomal-dominant polycystic kidney disease.
ROCK inhibitors modulate the physical properties and adipogenesis of 3D spheroids of human orbital fibroblasts in different manners.
Clinical characteristics of Slovenian pediatric patients with autosomal recessive polycystic kidney disease.
The ROCK inhibitor Y-27632 ameliorates blood-spinal cord barrier disruption by reducing tight junction protein degradation via the MYPT1-MLC2 pathway after spinal cord injury in rats.
Sub-centrosomal mapping identifies augmin-γTuRC as part of a centriole-stabilizing scaffold.
Rab-like small GTPases in the regulation of ciliary Bardet-Biedl syndrome (BBS)some transport.
DJ4 Targets the Rho-Associated Protein Kinase Pathway and Attenuates Disease Progression in Preclinical Murine Models of Acute Myeloid Leukemia.
A 7-year-old girl with renal medullary hyperechogenicity and hypertension: Answers.
Lysophosphatidic Acid Mediates Imiquimod-Induced Psoriasis-like Symptoms by Promoting Keratinocyte Proliferation through LPAR1/ROCK2/PI3K/AKT Signaling Pathway.
LncRNA KCNQ1OT1 promotes the development of diabetic nephropathy by regulating miR-93-5p/ROCK2 axis.
MAPK15 Controls Hedgehog Signaling in Medulloblastoma Cells by Regulating Primary Ciliogenesis.
Dynamic Changes of Brain Cilia Transcriptomes across the Human Lifespan.
Activation of mTOR mediates hyperglycemia-induced renal glomerular endothelial hyperpermeability via the RhoA/ROCK/pMLC signaling pathway.
Structural organization of the C1b projection within the ciliary central apparatus.
RhoA enhances osteosarcoma resistance to MPPa-PDT via the Hippo/YAP signaling pathway.
Sequential genetic testing of living related donors for inherited renal disease to promote informed choice and enhance safety of living donation.
NEK6 is an injury-responsive kinase cooperating with STAT3 in regulation of reactive astrogliosis.

Clin Case Rep. 2021 Oct;9(10): e04922

Celiac artery dissection in polycystic kidney disease.

Koichiro Yamamoto, Kou Hasegawa, Kosuke Oka, Fumio Otsuka.

  Autosomal-dominant polycystic kidney disease (ADPKD) is rarely complicated by celiac artery dissection. Dissection of the aorta and its major branches should be carefully differentiated in ADPKD patients with acute-onset abdominal pain.

Keywords:  autosomal‐dominant polycystic kidney disease; hypertension and mesenteric artery dissection

DOI:  https://doi.org/10.1002/ccr3.4922
Nat Genet. 2021 Oct 11.

Renal plasticity revealed through reversal of polycystic kidney disease in mice.

Ke Dong, Chao Zhang, Xin Tian, Daniel Coman, Fahmeed Hyder, Ming Ma, Stefan Somlo.

Initiation of cyst formation in autosomal dominant polycystic kidney disease (ADPKD) occurs when kidney tubule cells are rendered null for either PKD1 or PKD2 by somatic 'second hit' mutations. Subsequent cyst progression remodels the organ through changes in tubule cell shape, proliferation and secretion. The kidney develops inflammation and fibrosis. We constructed a mouse model in which adult inactivation of either Pkd gene can be followed by reactivation of the gene at a later time. Using this model, we show that re-expression of Pkd genes in cystic kidneys results in rapid reversal of ADPKD. Cyst cell proliferation is reduced, autophagy is activated and cystic tubules with expanded lumina lined by squamoid cells revert to normal lumina lined by cuboidal cells. Increases in inflammation, extracellular matrix deposition and myofibroblast activation are reversed, and the kidneys become smaller. We conclude that phenotypic features of ADPKD are reversible and that the kidney has an unexpected capacity for plasticity controlled at least in part by ADPKD gene function.

DOI: https://doi.org/10.1038/s41588-021-00946-4
Int J Mol Sci. 2021 Sep 29. pii: 10512. [Epub ahead of print]22(19):

Role of DNA-Dependent Protein Kinase in Mediating Cyst Growth in Autosomal Dominant Polycystic Kidney Disease.

Ashley N Chandra, Sayanthooran Saravanabavan, Gopala K Rangan.

  DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein involved in DNA damage response (DDR) signaling that may mediate kidney cyst growth in autosomal dominant polycystic kidney disease (ADPKD) due to its pleiotropic effects on proliferation and survival. To test this hypothesis, the expression of DNA-PK in human ADPKD and the in vitro effects of DNA-PK inhibition in a three-dimensional model of Madin-Darby Canine Kidney (MDCK) cyst growth and human ADPKD cells were assessed. In human ADPKD, the mRNA expression for all three subunits of the DNA-PK complex was increased, and using immunohistochemistry, the catalytic subunit (DNA-PKcs) was detected in the cyst lining epithelia of human ADPKD, in a focal manner. In vitro, NU7441 (a DNA-PK kinase inhibitor) reduced MDCK cyst growth by up to 52% after long-term treatment over 6-12 days. Although human ADPKD cell lines (WT9-7/WT9-12) did not exhibit synthetic lethality in response to DNA-PK kinase inhibition compared to normal human kidney cells (HK-2), the combination of low-dose NU7441 enhanced the anti-proliferative effects of sirolimus in WT9-7 and WT9-12 cells by 17 ± 10% and 11 ± 7%, respectively. In conclusion, these preliminary data suggest that DNA-PK mediates kidney cyst growth in vivo without a synthetically lethal interaction, conferring cell-specificity in human ADPKD cells. NU7441 enhanced the anti-proliferative effects of rapamycin complex 1 inhibitors, but the effect was modest.

Keywords:  DNA damage signaling; DNA-dependent protein kinase; double strand break; kidney cyst; kinase inhibitors; proliferation

DOI:  https://doi.org/10.3390/ijms221910512
Cell Death Dis. 2021 Oct 14. 12(10): 947

The tyrosine-kinase inhibitor Nintedanib ameliorates autosomal-dominant polycystic kidney disease.

Abeda Jamadar, Sreenath M Suma, Sijo Mathew, Timothy A Fields, Darren P Wallace, James P Calvet, Reena Rao.

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and is characterized by progressive growth of fluid-filled cysts. Growth factors binding to receptor tyrosine kinases (RTKs) stimulate cell proliferation and cyst growth in PKD. Nintedanib, a triple RTK inhibitor, targets the vascular endothelial growth-factor receptor (VEGFR), platelet-derived growth-factor receptor (PDGFR), and fibroblast growth-factor receptor (FGFR), and is an approved drug for the treatment of non-small-cell lung carcinoma and idiopathic lung fibrosis. To determine if RTK inhibition using nintedanib can slow ADPKD progression, we tested its effect on human ADPKD renal cyst epithelial cells and myofibroblasts in vitro, and on Pkd1f/fPkhd1Cre and Pkd1RC/RC, orthologous mouse models of ADPKD. Nintedanib significantly inhibited cell proliferation and in vitro cyst growth of human ADPKD renal cyst epithelial cells, and cell viability and migration of human ADPKD renal myofibroblasts. Consistently, nintedanib treatment significantly reduced kidney-to-body-weight ratio, renal cystic index, cystic epithelial cell proliferation, and blood-urea nitrogen levels in both the Pkd1f/fPkhd1Cre and Pkd1RC/RC mice. There was a corresponding reduction in ERK, AKT, STAT3, and mTOR activity and expression of proproliferative factors, including Yes-associated protein (YAP), c-Myc, and Cyclin D1. Nintedanib treatment significantly reduced fibrosis in Pkd1RC/RC mice, but did not affect renal fibrosis in Pkd1f/fPkhd1Cre mice. Overall, these results suggest that nintedanib may be repurposed to effectively slow cyst growth in ADPKD.

DOI: https://doi.org/10.1038/s41419-021-04248-9
FASEB Bioadv. 2021 Oct;3(10): 866-872

ROCK inhibitors modulate the physical properties and adipogenesis of 3D spheroids of human orbital fibroblasts in different manners.

Fumihito Hikage, Hanae Ichioka, Megumi Watanabe, Araya Umetsu, Hiroshi Ohguro, Yosuke Ida.

  To elucidate the pharmacological effects of Rho-associated coiled-coil containing protein kinase inhibitors (ROCK-is), ripasudil (Rip), Y27632, and KD025, on human orbital fatty tissue, the human orbital fibroblasts (HOFs) were three-dimensional (3D) cultured for 12 days. The effects of ROCK-is on the physical properties of the 3D-cultured HOF spheroids, including their sizes and physical stiffness, their adipogenesis by lipid staining, and the mRNA expression of adipogenesis-related genes, PPARγ and AP2, and extracellular matrix (ECM) including collagen (COL) 1, 4, and 6, and fibronectin were analyzed. A significant increase in the sizes, physical stiffness, lipid staining, and mRNA expression of adipogenesis-related genes, COL4 and COL6, and a decrease in COL1 expression were observed with adipogenesis (DIF+). In the presence of ROCK-is, such DIF+-induced effects were differently modulated as follows: (1) the sizes were not affected or significantly enhanced by Rip, Y27632, or KD025, (2) the physical stiffness was significantly decreased in Rip and Y27632, but was substantially increased in KD025, (3) the lipid staining was further enhanced or significantly suppressed by Rip, Y27632, or KD025, and both PPARγ and AP2 expression were significantly downregulated or upregulated by KD025 or Rip, and (4) Rip upregulated the expression of COL4, Y27632 upregulated the expression of COL1, COL4, and COL6, and KD025 upregulated the expression of COL1 and COL4. This study indicates that ROCK-is significantly and differently modulate physical properties of the 3D HOF spheroids as well as their adipogenesis.

Keywords:  ROCK; ROCK inhibitor; Rho kinase; human orbital fibroblasts (HOFs); three‐dimensional (3D) tissue culture

DOI:  https://doi.org/10.1096/fba.2021-00037
Clin Nephrol. 2021 Suppl;96(1):96(1): 56-61

Clinical characteristics of Slovenian pediatric patients with autosomal recessive polycystic kidney disease.

Anja Fon Gabršček, Anamarija Meglič, Gregor Novljan, Tanja Kersnik Levart, Rina Rus.

AIMS: Autosomal recessive polycystic kidney disease (ARPKD) is a rare inherited disease. We reviewed the clinical characteristics, management, and outcomes in Slovenian pediatric patients with ARPKD.MATERIALS AND METHODS: All patients with ARPKD who were treated at the Pediatric Nephrology Department of the University Children's Hospital in Ljubljana between 1980 and 2020 were included in the study. The data were assessed retrospectively by reviewing the patients' medical records and analyzed using descriptive statistics.
RESULTS: We included 13 patients, 6 boys and 7 girls. A prenatal diagnosis was established in 3 (23%) patients. In 4 (31%) patients, the diagnosis was confirmed within the first few days of life, while in 6 (46%) patients the disease manifested later during childhood. Four babies (31%) needed ventilatory support after birth. Arterial hypertension developed in all patients. Liver function was affected in 12 (92%) patients and was the predominant clinical concern in 2 of them. Two (15%) patients presented with end-stage renal disease (ESRD). Portal hypertension was found in 7 (54%) patients. Initial sonography revealed enlarged kidneys in 12 (92%) patients, hyperechoic kidneys or poor cortico-medullary differentiation in 10 (77%), and liver abnormalities in 5 (38%) patients. Unilateral nephrectomy was necessary before dialysis in 1 patient. Six (46%) patients started maintenance dialysis at an average age of 15.3 years. Kidney transplantation was performed in 2 (15%) and liver transplantation in 1 (8%) patient. Two (15%) patients died because of sepsis or respiratory failure.
CONCLUSION: ARPKD is a progressive disease leading to ESRD and renal replacement treatment in almost half of our patients. Our data confirm the phenotypic variability of ARPKD in Slovenian patients.

DOI: https://doi.org/10.5414/CNP96S10
Brain Res. 2021 Oct 09. pii: S0006-8993(21)00541-2. [Epub ahead of print]1773 147684

The ROCK inhibitor Y-27632 ameliorates blood-spinal cord barrier disruption by reducing tight junction protein degradation via the MYPT1-MLC2 pathway after spinal cord injury in rats.

Sheng Chang, Yang Cao.

  The blood-spinal cord barrier (BSCB) is a physiological barrier between the blood and spinal cord parenchyma. This study aims to determine whether Y-27632, a Rho-associated protein kinase (ROCK) inhibitor, can protect the BSCB using in vivo models. The Evans blue fluorescence assay was used to detect leakage of the BSCB. Western blotting was used to define alterations in ROCK-related and tight junction (TJ) protein expression. Immunofluorescence triple-staining was used to evaluate histologic alterations in TJs. Locomotor function was evaluated using the open-field test, the Basso-Beattie-Bresnahan score, and footprint analysis. Two peaks of BSCB leakage after spinal cord injury (SCI) occurred at 24 h and 5 days. The ROCK inhibitor reduced the BSCB leakage at the second peak after SCI. Moreover, the ROCK inhibitor ameliorated the integrity of the BSCB and improved motor function recovery after SCI by regulating the phosphorylation of myosin phosphatase subunit-1 (MYPT1) and cofilin. ROCK inhibitors might protect the BSCB, which provides a new strategy for transitioning SCI treatment from the bench to bedside.

Keywords:  Blood-spinal cord barrier; Evans blue; ROCK inhibitor; Spinal cord injury; Tight junction

DOI:  https://doi.org/10.1016/j.brainres.2021.147684
Nat Commun. 2021 Oct 15. 12(1): 6042

Sub-centrosomal mapping identifies augmin-γTuRC as part of a centriole-stabilizing scaffold.

Nina Schweizer, Laurence Haren, Ilaria Dutto, Ricardo Viais, Cristina Lacasa, Andreas Merdes, Jens Lüders.

Centriole biogenesis and maintenance are crucial for cells to generate cilia and assemble centrosomes that function as microtubule organizing centers (MTOCs). Centriole biogenesis and MTOC function both require the microtubule nucleator γ-tubulin ring complex (γTuRC). It is widely accepted that γTuRC nucleates microtubules from the pericentriolar material that is associated with the proximal part of centrioles. However, γTuRC also localizes more distally and in the centriole lumen, but the significance of these findings is unclear. Here we identify spatially and functionally distinct subpopulations of centrosomal γTuRC. Luminal localization is mediated by augmin, which is linked to the centriole inner scaffold through POC5. Disruption of luminal localization impairs centriole integrity and interferes with cilium assembly. Defective ciliogenesis is also observed in γTuRC mutant fibroblasts from a patient suffering from microcephaly with chorioretinopathy. These results identify a non-canonical role of augmin-γTuRC in the centriole lumen that is linked to human disease.

DOI: https://doi.org/10.1038/s41467-021-26252-5
FEBS J. 2021 Oct 16.

Rab-like small GTPases in the regulation of ciliary Bardet-Biedl syndrome (BBS)some transport.

Xiumin Yan, Yidong Shen.

  Primary cilia, microtubule-based hair-like structures protruding from most cells, contain membranes enriched in signaling molecules and function as sensory and regulatory organelles critical for development and tissue homeostasis. Intraflagellar transport (IFT), cilia-specific bidirectional transport, is required for the assembly, maintenance, and function of cilia. BBSome, the coat complex, acts as the adaptor between the IFT complex and membrane proteins and is therefore essential for establishing the specific compartmentalization of signaling molecules in the cilia. Recent findings have revealed that three ciliary Rab-like small GTPases, IFT27, IFT22, and Rabl2, play critical regulatory roles in ciliary BBSome transport. In this review, we provide an overview of these three Rab-like small GTPases and their relationship with BBSome.

Keywords:  BBSome; Bardet-Biedl syndrome; Cilia; IFT22; IFT27; Rab-like; Rabl2; small GTPases

DOI:  https://doi.org/10.1111/febs.16232
Cancers (Basel). 2021 Sep 29. pii: 4889. [Epub ahead of print]13(19):

DJ4 Targets the Rho-Associated Protein Kinase Pathway and Attenuates Disease Progression in Preclinical Murine Models of Acute Myeloid Leukemia.

Upendarrao Golla, Melanie A Ehudin, Charyguly Annageldiyev, Zheng Zeng, Diwakar Bastihalli Tukaramrao, Anna Tarren, Abhijit A Date, Irina Elcheva, Arthur Berg, Shantu Amin, Thomas P Loughran, Mark Kester, Dhimant Desai, Sinisa Dovat, David Claxton, Arati Sharma.

  The poor prognosis of acute myeloid leukemia (AML) and the highly heterogenous nature of the disease motivates targeted gene therapeutic investigations. Rho-associated protein kinases (ROCKs) are crucial for various actin cytoskeletal changes, which have established malignant consequences in various cancers, yet are still not being successfully utilized clinically towards cancer treatment. This work establishes the therapeutic activity of ROCK inhibitor (5Z)-2-5-(1H-pyrrolo[2,3-b]pyridine-3-ylmethylene)-1,3-thiazol-4(5H)-one (DJ4) in both in vitro and in vivo preclinical models of AML to highlight the potential of this class of inhibitors. Herein, DJ4 induced cytotoxic and proapoptotic effects in a dose-dependent manner in human AML cell lines (IC50: 0.05-1.68 μM) and primary patient cells (IC50: 0.264-13.43 μM); however, normal hematopoietic cells were largely spared. ROCK inhibition by DJ4 disrupts the phosphorylation of downstream targets, myosin light chain (MLC2) and myosin-binding subunit of MLC phosphatase (MYPT), yielding a potent yet selective treatment response at micromolar concentrations, from 0.02 to 1 μM. Murine models injected with luciferase-expressing leukemia cell lines subcutaneously or intravenously and treated with DJ4 exhibited an increase in overall survival and reduction in disease progression relative to the vehicle-treated control mice. Overall, DJ4 is a promising candidate to utilize in future investigations to advance the current AML therapy.

Keywords:  DJ4; Rho-associated protein kinase; acute myeloid leukemia; cell line-derived xenograft; preclinical AML murine model; primary human AML cells

DOI:  https://doi.org/10.3390/cancers13194889
Pediatr Nephrol. 2021 Oct 11.

A 7-year-old girl with renal medullary hyperechogenicity and hypertension: Answers.

İlknur Girişgen, Selcuk Yüksel, Furkan Ufuk, Taner Durak, Tülay Becerir.



Keywords:  ARPKD; Child; Genetic analysis; Hypertension; Medullary hyperechogenicity; Medullary nephrocalcinosis; Polycystic kidney disease

DOI:  https://doi.org/10.1007/s00467-021-05314-2
Int J Mol Sci. 2021 Oct 05. pii: 10777. [Epub ahead of print]22(19):

Lysophosphatidic Acid Mediates Imiquimod-Induced Psoriasis-like Symptoms by Promoting Keratinocyte Proliferation through LPAR1/ROCK2/PI3K/AKT Signaling Pathway.

Donghee Kim, Hyo-Jin Kim, Jin-Ok Baek, Joo-Young Roh, Hee-Sook Jun.

  Psoriasis is a chronic inflammatory skin disease. Recently, lysophosphatidic acid (LPA)/LPAR5 signaling has been reported to be involved in both NLRP3 inflammasome activation in macrophages and keratinocyte activation to produce inflammatory cytokines, contributing to psoriasis pathogenesis. However, the effect and molecular mechanisms of LPA/LPAR signaling in keratinocyte proliferation in psoriasis remain unclear. In this study, we investigated the effects of LPAR1/3 inhibition on imiquimod (IMQ)-induced psoriasis-like mice. Treatment with the LPAR1/3 antagonist, ki16425, alleviated skin symptoms in IMQ-induced psoriasis-like mouse models and decreased keratinocyte proliferation in the lesion. It also decreased LPA-induced cell proliferation and cell cycle progression via increased cyclin A2, cyclin D1, cyclin-dependent kinase (CDK)2, and CDK4 expression and decreased p27Kip1 expression in HaCaT cells. LPAR1 knockdown in HaCaT cells reduced LPA-induced proliferation, suppressed cyclin A2 and CDK2 expression, and restored p27Kip1 expression. LPA increased Rho-associated protein kinase 2 (ROCK2) expression and PI3K/AKT activation; moreover, the pharmacological inhibition of ROCK2 and PI3K/AKT signaling suppressed LPA-induced cell cycle progression. In conclusion, we demonstrated that LPAR1/3 antagonist alleviates IMQ-induced psoriasis-like symptoms in mice, and in particular, LPAR1 signaling is involved in cell cycle progression via ROCK2/PI3K/AKT pathways in keratinocytes.

Keywords:  Ki16425; keratinocyte; lysophosphatidic acid (LPA); proliferation; psoriasis

DOI:  https://doi.org/10.3390/ijms221910777
Diabetol Metab Syndr. 2021 Oct 15. 13(1): 108

LncRNA KCNQ1OT1 promotes the development of diabetic nephropathy by regulating miR-93-5p/ROCK2 axis.

Li Zhao, Huaqian Chen, Lin Wu, Zhengdong Li, Ren Zhang, Yan Zeng, Tao Yang, Hualing Ruan.

  BACKGROUND: Long non-coding RNAs (lncRNAs) have been reported to play vital roles in diabetic nephropathy (DN). The aim of this study was to explore the function of mechanism of lncRNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) in DN.METHODS: DN cell models were established using high glucose (HG) treatment in human glomerular mesangial cells (HGMC) and human renal glomerular endothelial cells (HRGEC). The expression levels of KCNQ1OT1, microRNA-93-5p (miR-93-5p), and Rho associated coiled-coil containing protein kinase 2 (ROCK2) mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell Counting Kit-8 (CCK-8) assay and flow cytometry were used to detect cell proliferation and apoptosis, respectively. ROCK2 and apoptosis/fibrosis-related protein levels were examined by western blot. The predicted interaction between miR-93-5p and KCNQ1OT1 or ROCK2 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay.
RESULTS: KCNQ1OT1 was upregulated in DN patients and DN cell models. KCNQ1OT1 knockdown inhibited cell proliferation and fibrosis and induced apoptosis in DN cell models. MiR-93-5p was a direct target of KCNQ1OT1, and miR-93-5p inhibition restored the KCNQ1OT1 knockdown-mediated effects on cell proliferation, fibrosis and apoptosis in DN cell models. In addition, ROCK2 was identified as a target of miR-93-5p, and miR-93-5p overexpression suppressed cell proliferation and fibrosis and accelerated apoptosis by targeting ROCK2 in DN cell models. Moreover, KCNQ1OT1 regulated ROCK2 expression by binding to miR-93-5p.
CONCLUSION: KCNQ1OT1 knockdown inhibited cell proliferation and fibrosis and induced apoptosis in DN by regulating miR-93-5p/ROCK2 axis, providing potential value for the treatment of DN.

Keywords:  Diabetic nephropathy; KCNQ1OT1; ROCK2; miR-93-5p

DOI:  https://doi.org/10.1186/s13098-021-00726-4
Cancers (Basel). 2021 Sep 29. pii: 4903. [Epub ahead of print]13(19):

MAPK15 Controls Hedgehog Signaling in Medulloblastoma Cells by Regulating Primary Ciliogenesis.

Silvia Pietrobono, Lorenzo Franci, Francesco Imperatore, Cristina Zanini, Barbara Stecca, Mario Chiariello.

  In medulloblastomas, genetic alterations resulting in over-activation and/or deregulation of proteins involved in Hedgehog (HH) signaling lead to cellular transformation, which can be prevented by inhibition of primary ciliogenesis. Here, we investigated the role of MAPK15 in HH signaling and, in turn, in HH-mediated cellular transformation. We first demonstrated, in NIH3T3 mouse fibroblasts, the ability of this kinase of controlling primary ciliogenesis and canonical HH signaling. Next, we took advantage of transformed human medulloblastoma cells belonging to the SHH-driven subtype, i.e., DAOY and ONS-76 cells, to ascertain the role for MAPK15 in HH-mediated cellular transformation. Specifically, medullo-spheres derived from these cells, an established in vitro model for evaluating progression and malignancy of putative tumor-initiating medulloblastoma cells, were used to demonstrate that MAPK15 regulates self-renewal of these cancer stem cell-like cells. Interestingly, by using the HH-related oncogenes SMO-M2 and GLI2-DN, we provided evidences that disruption of MAPK15 signaling inhibits oncogenic HH overactivation in a specific cilia-dependent fashion. Ultimately, we show that pharmacological inhibition of MAPK15 prevents cell proliferation of SHH-driven medulloblastoma cells, overall suggesting that oncogenic HH signaling can be counteracted by targeting the ciliary gene MAPK15, which could therefore be considered a promising target for innovative "smart" therapies in medulloblastomas.

Keywords:  GLI1; MAP Kinases; cancer stem cells; medullo-spheres; primary cilia

DOI:  https://doi.org/10.3390/cancers13194903
Int J Mol Sci. 2021 Sep 27. pii: 10387. [Epub ahead of print]22(19):

Dynamic Changes of Brain Cilia Transcriptomes across the Human Lifespan.

Siwei Chen, Wedad Alhassen, Roudabeh Vakil Monfared, Benjamin Vachirakorntong, Surya M Nauli, Pierre Baldi, Amal Alachkar.

  Almost all brain cells contain primary cilia, antennae-like microtubule sensory organelles, on their surface, which play critical roles in brain functions. During neurodevelopmental stages, cilia are essential for brain formation and maturation. In the adult brain, cilia play vital roles as signaling hubs that receive and transduce various signals and regulate cell-to-cell communications. These distinct roles suggest that cilia functions, and probably structures, change throughout the human lifespan. To further understand the age-dependent changes in cilia roles, we identified and analyzed age-dependent patterns of expression of cilia's structural and functional components across the human lifespan. We acquired cilia transcriptomic data for 16 brain regions from the BrainSpan Atlas and analyzed the age-dependent expression patterns using a linear regression model by calculating the regression coefficient. We found that 67% of cilia transcripts were differentially expressed genes with age (DEGAs) in at least one brain region. The age-dependent expression was region-specific, with the highest and lowest numbers of DEGAs expressed in the ventrolateral prefrontal cortex and hippocampus, respectively. The majority of cilia DEGAs displayed upregulation with age in most of the brain regions. The transcripts encoding cilia basal body components formed the majority of cilia DEGAs, and adjacent cerebral cortices exhibited large overlapping pairs of cilia DEGAs. Most remarkably, specific α/β-tubulin subunits (TUBA1A, TUBB2A, and TUBB2B) and SNAP-25 exhibited the highest rates of downregulation and upregulation, respectively, across age in almost all brain regions. α/β-tubulins and SNAP-25 expressions are known to be dysregulated in age-related neurodevelopmental and neurodegenerative disorders. Our results support a role for the high dynamics of cilia structural and functional components across the lifespan in the normal physiology of brain circuits. Furthermore, they suggest a crucial role for cilia signaling in the pathophysiological mechanisms of age-related psychiatric/neurological disorders.

Keywords:  brain; cilia; human; lifespan; linear regression; transcriptome

DOI:  https://doi.org/10.3390/ijms221910387
Diabetol Metab Syndr. 2021 Oct 09. 13(1): 105

Activation of mTOR mediates hyperglycemia-induced renal glomerular endothelial hyperpermeability via the RhoA/ROCK/pMLC signaling pathway.

Xiaolin Chen, Jianhui Chen, Xianfan Li, Zengpu Yu.

  OBJECTIVE: Hyperglycemia is associated with albuminuria and renal glomerular endothelial dysfunction in patients with diabetic nephropathy. The mTOR and RhoA/ROCK signaling pathways are involved in glomerular filtration barrier (GFB) regulation, but their role in high glucose (HG)-induced GFB dysfunction in human renal glomerular endothelial cells (HRGECs) has not been investigated. This study aimed to investigate the mechanisms of HG-induced GFB dysfunction in vitro.MATERIALS AND METHODS: HRGECs were cultured in vitro and exposed to HG. The horseradish peroxidase-albumin leakage and transendothelial electrical resistance of the endothelial monolayer were measured after HG treatment with or without rapamycin preincubation. A fluorescence probe was used to study the distribution of F-actin reorganization. The phosphorylation levels of myosin light chain (MLC) and mTOR were measured via western blotting. RhoA activity was evaluated via GTPase activation assay. The effects of blocking mTOR or the RhoA/ROCK pathway on endothelial permeability and MLC phosphorylation under HG conditions were observed.
RESULTS: HG exposure induced F-actin reorganization and increased MLC phosphorylation, leading to EC barrier disruption. This effect was attenuated by treatment with rapamycin or Y-27632. Phospho-MLC (pMLC) activation in HRGECs was mediated by RhoA/ROCK signaling. mTOR and RhoA/ROCK inhibition or knockdown attenuated pMLC activation, F-actin reorganization and barrier disruption that occurred in response to HG exposure.
CONCLUSIONS: Our results revealed that HG stimulation upregulated RhoA expression and activity through an mTOR-dependent pathway, leading to MLC-mediated endothelial cell cytoskeleton rearrangement and glomerular endothelial barrier dysfunction.

Keywords:  Glomerular endothelial cell; High glucose; Myosin light chain; Permeability; RhoA/ROCK

DOI:  https://doi.org/10.1186/s13098-021-00723-7
J Cell Sci. 2021 Oct 15. pii: jcs.254227. [Epub ahead of print]

Structural organization of the C1b projection within the ciliary central apparatus.

Kai Cai, Yanhe Zhao, Lei Zhao, Nhan Phan, Yuqing Hou, Xi Cheng, George B Witman, Daniela Nicastro.

  '9+2' motile cilia contain 9 doublet microtubules and a central apparatus (CA) composed of two singlet microtubules with associated projections. The CA plays crucial roles in regulating ciliary motility. Defects in CA assembly or function usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of most CA-projections remain largely unknown. Here, we combined genetic, proteomic, and cryo-electron tomographic approaches to compare the CA of wild-type Chlamydomonas with those of three CA-mutants. Our results show that two proteins, FAP42 and FAP246, are localized to the L-shaped C1b-projection of the CA, where they interact with the candidate CA-protein FAP413. FAP42 is a large protein that forms the peripheral 'beam' of the C1b-projection, and the FAP246-FAP413 subcomplex serves as the 'bracket' between the beam (FAP42) and the C1b 'pillar' that attaches the projection to the C1-microtubule. The FAP246-FAP413-FAP42 complex is essential for stable assembly of the C1b, C1f and C2b-projections, and loss of these proteins leads to ciliary motility defects.

Keywords:  Axoneme; Central pair complex; Cryo-electron tomography; Flagella; Subtomogram averaging

DOI:  https://doi.org/10.1242/jcs.254227
Cell Biosci. 2021 Oct 09. 11(1): 179

RhoA enhances osteosarcoma resistance to MPPa-PDT via the Hippo/YAP signaling pathway.

Fangbiao Zhan, Tao He, Zhiyu Chen, Qiang Zuo, Yang Wang, Qiaochu Li, Shenxi Zhong, Yunsheng Ou.

  BACKGROUND: Osteosarcoma (OS) is the most prevalent primary bone malignancy affecting adolescents, yet the emergence of chemoradiotherapeutic resistance has limited efforts to cure affected patients to date. Pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPa-PDT) is a recently developed, minimally invasive treatment for OS that is similarly constrained by such therapeutic resistance. This study sought to explore the mechanistic basis for RhoA-activated YAP1 (YAP)-mediated resistance in OS.METHODS: The relationship between YAP expression levels and patient prognosis was analyzed, and YAP levels in OS cell lines were quantified. Immunofluorescent staining was used to assess YAP nuclear translocation. OS cell lines (HOS and MG63) in which RhoA and YAP were knocked down or overexpressed were generated using lentiviral vectors. CCK-8 assays were used to examine OS cell viability, while the apoptotic death of these cells was monitored via Hoechst staining, Western blotting, and flow cytometry. Tumor-bearing nude mice were additionally used to assess the relationship between lentivirus-mediated alterations in RhoA expression and MPPa-PDT treatment outcomes. TUNEL and immunohistochemical staining approaches were leveraged to assess apoptotic cell death in tissue samples.
RESULTS: OS patients exhibited higher levels of YAP expression, and these were correlated with a poor prognosis. MPPa-PDT induced apoptosis in OS cells, and such MPPa-PDT-induced apoptosis was enhanced following YAP knockdown whereas it was suppressed by YAP overexpression. RhoA and YAP expression levels were positively correlated in OS patients, and both active and total RhoA protein levels rose in OS cells following MPPa-PDT treatment. When RhoA was knocked down, levels of unphosphorylated YAP and downstream target genes were significantly reduced, while RhoA/ROCK2/LIMK2 pathway phosphorylation was suppressed, whereas RhoA overexpression resulted in the opposite phenotype. MPPa-PDT treatment was linked to an increase in HMGCR protein levels, and the inhibition of RhoA or HMGCR was sufficient to suppress RhoA activity and to decrease the protein levels of YAP and its downstream targets. Mevalonate administration partially reversed these reductions in the expression of YAP and YAP target genes. RhoA knockdown significantly enhanced the apoptotic death of OS cells in vitro and in vivo following MPPa-PDT treatment, whereas RhoA overexpression had the opposite effect.
CONCLUSIONS: These results suggest that the mevalonate pathway activates RhoA, which in turn activates YAP and promotes OS cell resistance to MPPa-PDT therapy. Targeting the RhoA/ROCK2/LIMK2/YAP pathway can significantly improve the efficacy of MPPa-PDT treatment for OS.

Keywords:  Apoptosis; MPPa-PDT; Mevalonate; Osteosarcoma; RhoA; YAP

DOI:  https://doi.org/10.1186/s13578-021-00690-6
Transpl Int. 2021 Oct 10.

Sequential genetic testing of living related donors for inherited renal disease to promote informed choice and enhance safety of living donation.

Christie P Thomas, Sonali Gupta, Margaret E Freese, Kanwaljit K Chouhan, Maisie I Dantuma, Danniele G Holanda, Daniel A Katz, Benjamin W Darbro, M Adela Mansilla, Richard J Smith.

  Living kidney donors (LKDs) with a family history of renal disease are at risk of kidney disease as compared to LKDs without such history suggesting that some LKDs may be pre-symptomatic for monogenic kidney disease. LKDs with related transplant candidates whose kidney disease was considered genetic in origin were selected for genetic testing. In each case, the transplant candidate was first tested to verify the genetic diagnosis. A genetic diagnosis was confirmed in 12 of 24 transplant candidates (ADPKD-PKD1: 6, ALPORT-COL4A3: 2, ALPORT-COL4A5: 1: nephronophthisis-SDCCAG8: 1; CAKUT-HNF1B and ADTKD-MUC1: 1 each) and 2 had variants of unknown significance (VUS) in phenotype-relevant genes. Focused genetic testing was then done in 20 of 34 LKDs. 12 LKDs screened negative for the familial variant and were permitted to donate; 7 screened positive and were counseled against donation. One, the heterozygous carrier of a recessive disorder was also cleared. 6 of 7 LKDs with a family history of ADPKD were under 30 yr and in 5, by excluding ADPKD, allowed donation to safely proceed. The inclusion of genetic testing clarified the diagnosis in recipient candidates, improving safety or informed decision making in LKDs.

Keywords:  Alport nephropathy; Chromosomal microarray; Genetic testing; Next-Generation Sequencing; Polycystic Disease; multiplex ligation-dependent probe amplification

DOI:  https://doi.org/10.1111/tri.14133
Glia. 2021 Oct 13.

NEK6 is an injury-responsive kinase cooperating with STAT3 in regulation of reactive astrogliosis.

Ying Yu, Tianjin Shen, Xiaoling Zhong, Lei-Lei Wang, Wenjiao Tai, Yuhua Zou, Jun Qin, Zhaohui Zhang, Chun-Li Zhang.

  In response to brain injury, resident astrocytes become reactive and play dynamic roles in neural repair and regeneration. The signaling pathways underlying such reactive astrogliosis remain largely unclear. We here show that NEK6, a NIMA-related serine/threonine protein kinase, is rapidly induced following pathological stimulations and plays critical roles in reactive astrogliosis. Enhanced NEK6 expression promotes reactive astrogliosis and exacerbates brain lesions; and conversely, NEK6 downregulation dampens injury-induced astrocyte reactivity and reduces lesion size. Mechanistically, NEK6 associates with and phosphorylates STAT3. Kinase activity of NEK6 is required for induction of GFAP and PCNA, markers of reactive astrogliosis. Interestingly, NEK6 is also localized in the nucleus and binds to STAT3-responsive genomic elements in astrocytes. These results indicate that NEK6 constitutes a molecular target for the regulation of reactive astrogliosis.

Keywords:  NEK6; STAT3; glial scar; neuroinflammation; reactive astrogliosis; traumatic brain injury

DOI:  https://doi.org/10.1002/glia.24104