Cells Sense Changes Research Articles

Abstract 01: Spatial Transcriptomics Reveals How Smooth Muscle Cells Transform to Renin-Expressing Cells During Homeostatic Challenge

Introduction: Juxtaglomerular (JG) cells sense changes in blood pressure through communication with neighboring cells, and/or the sympathetic nervous system upon which they release renin to maintain homeostasis. To meet the demands for circulating renin when homeostasis is threatened, smooth muscle cells (SMCs) of the renal arterioles are transformed to renin-synthesizing cells. Discriminating transformed cells versus JG cells using sorting and scRNA-seq is cumbersome, limiting our ability to explore the events underlying such transformation. Therefore, there is a need to study these cells in situ to assign and determine which epigenomic and cell communication pathways regulate renin expression and SMC transformation. Hypothesis: Transformation of SMCs to renin-expressing cells during homeostatic stress requires specific signals from neighboring cells in addition to intrinsic transcriptomic changes. Methods: A pilot study was performed using two three-month-old C57BL/6 mice fed either a normal diet, or a low-salt diet with the RAS inhibitor, captopril, to induce homeostatic stress. After one week, the kidneys were harvested and prepared for the 10X Genomics Visium platform. Sequencing results were preprocessed using the Space Ranger pipeline (v. 2.0.1) with the mouse mm10 genome prior to downstream analysis using the R packages Seurat, Signac, and CellChat. Results: Homeostatic challenge significantly increased Ren1 expression ( p Holm-adj. =4.63x10 -10 ) and led to more and stronger cellular interactions. Interstitial cells and JG cells contributed the largest percent increase in the observed signaling changes (6.5% and 4.5% respectively). Increased signals for collagen, amyloid precursor protein, osteopontin, laminin, thrombospondin 1, vascular endothelial growth factor, and growth arrest specific pathways drove changes between native and transformed renin-expressing cells. In addition to the established JG cell markers Ren1 and Akr1b7 , transformed cells had significantly increased expression ( p Bonferroni-adj < 0.01) of Hmgcs2 , Cyp4a14 , Suox , Acta2 , Slc2a5 , Ptp4a3 , Sncg , and Rad21 . Conclusions: In conclusion, spatial transcriptomics revealed the evolution of SMC transformation into renin phenotypes by uncovering pathways and genes understudied in the context of renin regulation. Future work will leverage higher resolution spatial technology and further explore the contribution of identified pathways to renin regulation and SMC transformation.

Studies on Membrane Potential and Ca2+ Oscillations in Rat Carotid Body Type I Cells

Carotid body (CB) glomus cells sense changes in arterial O2 pressure (PO2) and pH, and adjust the secretory and carotid sinus nerve activity and ventilation to help maintain normal levels of blood PO2 and pH. Our recent studies showed that isolated CB cell clusters perfused with physiological buffer solution generate spontaneous Ca2+ oscillations at low frequency in normoxia. Mild and moderate levels of hypoxia (2-5%O2) and acidosis (pHo7.2-7.3) increased the frequency and amplitude of Ca2+ oscillations. Inhibitors of voltage-dependent Ca2+ channels and removal of external Ca2+ abolished Ca2+ oscillations, indicating that Ca2+ influx was critical for generating Ca2+ oscillations. To better understand the phenomenon of Ca2+oscillations in glomus cells, we examined the potential role of oscillations in cell membrane potential (Em) in CB cells in triggering Ca2+ influx and Ca2+ oscillations. Using the cell-attached patch configuration, we assessed cell Em by recording TASK single channels, because a linear relationship exists between TASK amplitude and cell Em. Recording of TASK in CB cells in normoxia showed that many CB cells exhibit oscillations in TASK amplitude, indicating the presence of spontaneous oscillations in cell Em. The oscillation frequency of cell Em was similar to that of Ca2+ oscillations. Oscillations in TASK single channel amplitude were blocked by nifedipine (Ca2+ channel antagonist), by removal of extracellular Ca2+, and by 2-APB (an inhibitor of ER Ca2+ channel and store-operated Ca2+ entry). Mild hypoxia increased the frequency of oscillations of TASK amplitude, indicating that mild hypoxia increased the frequency of cell Em oscillations. These findings suggest that cell Em oscillations (that open voltage-dependent Ca2+ channels and increase Ca2+ influx) are an integral component of the cellular signaling mechanism by which Ca2+ oscillations are produced in CB cells. This work was funded by National Institutes of Health (NIH) grants to D.K. (HL111497) and C.W. (HL142906), and an award from Rosalind Franklin University of Medicine and Science. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acid-Sensitive Outwardly Rectifying Cl- Current in OV2944 Mouse Ovarian Cancer Cells.

Extracellular acidic conditions impair cellular activities; however, some cancer cells drive cellular signaling to adapt to the acidic environment. It remains unclear how ovarian cancer cells sense changes in extracellular pH. This study was aimed at characterizing acid-inducible currents in an ovarian cancer cell line and evaluating the involvement of these currents in cell viability. The biophysical and pharmacological properties of membrane currents in OV2944, a mouse ovarian cancer cell line, were studied using the whole-cell configuration of the patch-clamp technique. Viability of this cell type in acidic medium was evaluated using the MTT assay. OV2944 had significant acid-sensitive outwardly rectifying (ASOR) Cl- currents at a pH50 of 5.3. The ASOR current was blocked by pregnenolone sulfate (PS), a steroid ion channel modulator that blocks the ASOR channel as one of its targets. The viability of the cells was reduced after exposure to an acidic medium (pH 5.3)but was slightly restored upon PS administration. These results offer first evidence for the presence of ASOR Cl- channel in ovarian cancer cells and indicate its involvement in cell viability under acidic environment.

MPK12 in stomatal CO2 signaling: function beyond its kinase activity.

Protein phosphorylation is a major molecular switch involved in the regulation of stomatal opening and closure. Previous research defined interaction between MAP kinase 12 and Raf-like kinase HT1 as a required step for stomatal movements caused by changes in CO2 concentration. However, whether MPK12 kinase activity is required for regulation of CO2 -induced stomatal responses warrants in-depth investigation. We apply genetic, biochemical, and structural modeling approaches to examining the noncatalytic role of MPK12 in guard cell CO2 signaling that relies on allosteric inhibition of HT1. We show that CO2 /HCO3 - -enhanced MPK12 interaction with HT1 is independent of its kinase activity. By analyzing gas exchange of plant lines expressing various kinase-dead and constitutively active versions of MPK12 in a plant line where MPK12 is deleted, we confirmed that CO2 -dependent stomatal responses rely on MPK12's ability to bind to HT1, but not its kinase activity. We also demonstrate that purified MPK12 and HT1 proteins form a heterodimer in the presence of CO2 /HCO3 - and present structural modeling that explains the MPK12:HT1 interaction interface. These data add to the model that MPK12 kinase-activity-independent interaction with HT1 functions as a molecular switch by which guard cells sense changes in atmospheric CO2 concentration.

Farnesoid-X receptor as a therapeutic target for inflammatory bowel disease and colorectal cancer.

Farnesoid-X receptor (FXR), as a nuclear receptor activated by bile acids, is a vital molecule involved in bile acid metabolism. Due to its expression in immune cells, FXR has a significant effect on the function of immune cells and the release of chemokines when immune cells sense changes in bile acids. In addition to its regulation by ligands, FXR is also controlled by post-translational modification (PTM) activities such as acetylation, SUMOylation, and methylation. Due to the high expression of FXR in the liver and intestine, it significantly influences intestinal homeostasis under the action of enterohepatic circulation. Thus, FXR protects the intestinal barrier, resists bacterial infection, reduces oxidative stress, inhibits inflammatory reactions, and also acts as a tumor suppressor to impair the multiplication and invasion of tumor cells. These potentials provide new perspectives on the treatment of intestinal conditions, including inflammatory bowel disease (IBD) and its associated colorectal cancer (CRC). Moreover, FXR agonists on the market have certain organizational heterogeneity and may be used in combination with other drugs to achieve a greater therapeutic effect. This review summarizes current data on the role of FXR in bile acid metabolism, regulation of immune cells, and effects of the PTM of FXR. The functions of FXR in intestinal homeostasis and potential application in the treatment of IBD and CRC are discussed.

'Neighbourhood watch' model: embryonic epiblast cells assess positional information in relation to their neighbours.

ABSTRACTIn many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos, including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name the ‘neighbourhood watch’ model) illuminates the classic ‘French Flag Problem’ and how positional information is interpreted by a sheet of cells in a large developing system.

Substrate Stiffness Drives Epithelial to Mesenchymal Transition and Proliferation through the NEAT1-Wnt/β-Catenin Pathway in Liver Cancer.

Background: Extracellular matrix (ECM)-derived mechanical stimuli regulate many cellular processes and phenotypes through mechanotransduction signaling pathways. Substrate stiffness changes cell phenotypes and promotes angiogenesis, epithelial to mesenchymal transition (EMT), and metastasis in tumors. Enhanced liver tissue matrix stiffness plays a crucial role in the tumorigenesis and malignant development of liver cancer and is associated with unfavorable survival outcomes. However, how liver cancer cells sense changes in ECM stiffness and the underlying molecular mechanisms are largely unknown. Methods: Seeding HepG2 cells on the micropillar gels, HepG2 cells were assessed for responsiveness to mechanotransduction using Western blot and immunofluorescence. Conclusions: We found that higher substrate stiffness dramatically enhanced malignant cell phenotypes and promoted G1/S transition in HepG2 cells. Furthermore, nuclear paraspeckle assembly transcript 1 (NEAT1) was identified as a matrix stiffness-responsive long non-coding RNA (lncRNA) regulating proliferation and EMT in response to increasing matrix stiffness during the progression of HepG2 cells towards liver cancer phenotypes. Higher matrix stiffness contributed to enhancing NEAT1 expression, which activated the WNT/β-catenin pathway. β-catenin translocates and enters the nucleus and the EMT transcription factor zinc finger E-box binding homeobox 1 (ZEB1) was upregulated to trigger EMT. Additionally, the proteins required for matrix stiffness-induced proliferation and resistance were strikingly upregulated in HepG2 cells. Therefore, our findings provide evidence that ECM-derived mechanical signals regulate cell proliferation and drive EMT through a NEAT1/WNT/β-catenin mechanotransduction pathway in the tumor microenvironment of liver cancer.

Melatonin attenuates low shear stress-induced pyroptosis and endothelial cell dysfunction via the RORα/miR-223/STAT-3 signalling pathway.

Endothelial cells sense changes in blood flow shear stress and affect the progression of atherosclerotic plaques. Pyroptosis is an inflammatory form of cell death and has been implicated in cardiovascular diseases. Melatonin and its nuclear receptor retinoid-related orphan receptor α (RORα) have protective effects on the development of atherosclerosis. To date, whether melatonin can prevent endothelial cell pyroptosis and dysfunction in pathological shear stress remains unclear. In the present study, human umbilical vein endothelial cells (ECs) were cultured under low shear stress conditions (5 dyne/cm2) for 24 h and treated with or without melatonin (2 µmol/l). The binding sites of the microRNA (miR)-223 promoter and RORα were predicted using the JASPAR website. Expression of pyroptosis-related proteins, including cleaved N-terminal gasdermin D, caspase-1, intercellular adhesion molecule 1 (ICAM-1) and nitric oxide (NO) were assessed. The results indicated that low shear stress increased pyroptosis and ICAM-1 expression, whereas it decreased NO levels. Melatonin alleviated pyroptosis and ICAM-1 expression and increased the production of NO in ECs. Further assessment revealed that low-level shear stress decreased RORα protein and mRNA expression, whereas melatonin would bind to RORα and thereby promoted miR-223 transcription in ECs. The present study also identified signal transducer and activator of transcription 3 (STAT-3) as a potential target gene of miR-223-3p. When transfected with miR-223 inhibitor, ECs up-regulated the expression of pyroptosis-related proteins and ICAM-1, and down-regulated NO levels. By contrast, silencing STAT-3 expression diminished the protective effect of miR-223. These results indicated that melatonin prevented ECs from undergoing pyroptosis and alleviated dysfunction via the RORα/miR-223/STAT-3 signalling pathway. This information could aid in the development of novel therapeutic approaches and provide new insights into atherosclerosis.

Architecture and Assembly of the Bacterial Flagellar Motor Complex.

One of the central systems responsible for bacterial motility is the flagellum. The bacterial flagellum is a macromolecular protein complex that is more than five times the cell length. Flagella-driven motility is coordinated via a chemosensory signal transduction pathway, and so bacterial cells sense changes in the environment and migrate towards more desirable locations. The flagellum of Salmonella enterica serovar Typhimurium is composed of a bi-directional rotary motor, a universal joint and a helical propeller. The flagellar motor, which structurally resembles an artificial motor, is embedded within the cell envelop and spins at several hundred revolutions per second. In contrast to an artificial motor, the energy utilized for high-speed flagellar motor rotation is the inward-directed proton flow through a transmembrane proton channel of the stator unit of the flagellar motor. The flagellar motor realizes efficient chemotaxis while performing high-speed movement by an ingenious directional switching mechanism of the motor rotation. To build the universal joint and helical propeller structures outside the cell body, the flagellar motor contains its own protein transporter called a type III protein export apparatus. In this chapter we summarize the structure and assembly of the Salmonella flagellar motor complex.

Mast cell disorders: From infancy to maturity.

Mast cells are typically linked to immediate hypersensitivity and anaphylaxis. This review looks beyond this narrow role, focusing on how these cells have evolved and diversified via natural selection promoting serine protease gene duplication, augmenting their innate host defense function against helminths and snake envenomation. Plasticity of mast cell genes has come at a price. Somatic activating mutations in the mast cell growth factor KIT gene cause cutaneous mastocytosis in young children and systemic mastocytosis with a more guarded prognosis in adults who may also harbor other gene mutations with oncogenic potential as they age. Allelic TPSAB1 gene duplication associated with higher basal mast cell tryptase is possibly one of the commonest autosomal dominantly inherited multi-system diseases affecting the skin, gastrointestinal tract, circulation and musculoskeletal system. Mast cells are also establishing a new-found importance in severe asthma, and in remodeling of blood vessels in cancer and atherosclerotic vascular disease. Furthermore, recent evidence suggests that mast cells sense changes in oxygen tension, particularly in neonates, and that subsequent degranulation may contribute to common lung, eye, and brain diseases of prematurity classically associated with hypoxic insults. One hundred and forty years since Paul Ehrlich's initial description of "mastzellen," this review collates and highlights the complex and diverse roles that mast cells play in health and disease.

Mechanisms Underlying Freezing and Desiccation Tolerance in Bryophytes.

Bryophytes are small land plants that have many morphological and physiological features different from vascular plants. With distinct water relations of bryophytes, many bryophyte species exhibit high degrees of tolerance to freezing and desiccation. The tolerance is sustained by the constitutive repair mechanism and the inducible mechanism regulated by environmental signals that provoke specific responses within the cells. Bryophyte cells sense changes in environmental conditions such as decreases in osmotic potential and temperature and that some responses are likely to be mediated by the stress hormone, abscisic acid. Due to their simple structures and high degrees of dehydration tolerance, bryophytes are useful for physiological studies on abiotic stress response and also for analysis of signal sensing and transduction of environmental signals. Furthermore, the basal phylogenetic position of bryophytes in land plants provides many insights into the evolutionary events for conquest of land by the ancestors of plants and subsequent diversification of species as well as their survival strategies in the terrestrial environment.

Nutrient sensing in pancreatic islets: lessons from congenital hyperinsulinism and monogenic diabetes.

Pancreatic beta cells sense changes in nutrients during the cycles of fasting and feeding and release insulin accordingly to maintain glucose homeostasis. Abnormal beta cell nutrient sensing resulting from gene mutations leads to hypoglycemia or diabetes. Glucokinase (GCK) plays a key role in beta cell glucose sensing. As one form of congenital hyperinsulinism (CHI), activating mutations of GCK result in a decreased threshold for glucose-stimulated insulin secretion and hypoglycemia. In contrast, inactivating mutations of GCK result in diabetes, including a mild form (MODY2) and a severe form (permanent neonatal diabetes mellitus (PNDM)). Mutations of beta cell ion channels involved in insulin secretion regulation also alter glucose sensing. Activating or inactivating mutations of ATP-dependent potassium (KATP ) channel genes result in severe but completely opposite clinical phenotypes, including PNDM and CHI. Mutations of the other ion channels, including voltage-gated potassium channels (Kv 7.1) and voltage-gated calcium channels, also lead to abnormal glucose sensing and CHI. Furthermore, amino acids can stimulate insulin secretion in a glucose-independent manner in some forms of CHI, including activating mutations of the glutamate dehydrogenase gene, HDAH deficiency, and inactivating mutations of KATP channel genes. These genetic defects have provided insight into a better understanding of the complicated nature of beta cell fuel-sensing mechanisms.

Soft matrices inhibit cell proliferation and inactivate the fibrotic phenotype of deep endometriotic stromal cells in vitro.

Can deep infiltrating endometriotic stromal cells (DES) sense changes in extracellular matrix (ECM) stiffness and respond to them? Soft matrices inhibit cell proliferation and inactivate the fibrotic phenotype of DES in vitro. Deep infiltrating endometriosis (DIE) is characterized histologically by dense fibrous tissue. Tissue stiffening is a hallmark of fibrosis. Studies show that matrix stiffness is involved in the progression of numerous diseases, including cancer and fibrosis. However, no studies to date have investigated whether tissue stiffening could influence cell behavior in DIE. Previous in vitro studies typically analyzed cells grown on rigid plastic or glass substrates with stiffness in the gigapascal (gPa) range, which is much stiffer than that occurring in vivo. To investigate how changes in ECM stiffness affect the behavior of DES, it is critical to model in vivo tissue compliance conditions in vitro. For this laboratory study, paired endometrial and endometriotic samples from 40 patients who had histological evidence of DIE and endometrial samples from 23 patients without endometriosis were analyzed (uterine fibroma: n = 10, tubal infertility: n = 13). All participants were 20-37 years old and had regular menstrual cycles of 26-32 days. The abundance of F-actin, alpha smooth muscle actin (αSMA), Ki67, and procollagen type I in DES and endometrial stromal cells (EES) on polyacrylamide gel substrates of varying stiffness (2, 4, 8, 16 and/or 30 kPa) was determined by immunofluorescence confocal microscopy. mRNA level of type I collagen, matrix metalloproteinase-1 (MMP-1), MMP-14 and cyclin D1 was measured by real-time PCR. The cellular proliferation index (CPI), assessed as the percentage of Ki67-positive cells among the total number of nuclei stained by 4',6-diamidino-2-phenylindole (DAPI) was determined. Increased matrix stiffness induced F-actin stress fiber formation in both EES and DES, whereas αSMA-containing stress fibers were induced only in DES. Furthermore, increased stiffness increased the CPI in both EES (16 or 30 kPa versus 2 kPa, P < 0.05) and DES (16 or 30 kPa versus 2, 4 or 8 kPa, P < 0.05). Increased stiffness increased the percentage of procollagen I-positive cells as well as mRNA levels of type I collagen in both EES and DES in a matrix stiffness-dependent manner (2, 8 and 30 kPa) (P < 0.05). Increased stiffness also increased MMP-14 mRNA levels in EES (30 versus 2 kPa, P < 0.05), but decreased MMP-1 mRNA levels in DES in a matrix stiffness-dependent manner (2, 8 and 30 kPa; P < 0.05). Treatment with transforming growth factor (TGF)-β1 further increased type I collagen mRNA levels in both EES and DES when compared with cells grown on a substrate of the same stiffness (2, 8 or 30 kPa, with versus without TGF-β1, P < 0.05). Treatment with TGF-β1 also increased MMP-1 (8 or 30 kPa, P < 0.05 versus no TGF-β1) and MMP-14 mRNA levels (2, 8 or 30 kPa, P < 0.05 versus no TGF-β1) in EES, but decreased MMP-1 mRNA levels (2, 8 or 30 kPa, P < 0.05 versus no TGF-β1) in DES. On a soft substrate (2 kPa), both EES and DES exhibited a small rounded morphology with diffuse labeling for F-actin. No F-actin-positive stress fibers were observed in either EES or DES grown on 2 kPa substrates. There were more Ki67-positive EES when grown on 2, 4 or 8 kPa compared with Ki67-positive DES (P < 0.05). A tremendous gap exists between the present in vitro model and in vivo deep endometriotic tissues. Cell culture systems that more closely mimic the cellular complexity typical of in vivo endometriotic tissues are required to develop novel strategies for treatment of DIE. A disadvantage of polyacrylamide is its cytotoxicity but in the two-dimensional culture models used here, where cells are seeded above the polyacrylamide gel, this should not have a major impact. Finally, the soft substrates we used in vitro (2 and 4 kPa) may represent the elasticity of the endometrium in vivo, however, currently there are no data regarding tissue stiffness in DIE in vivo. Hormonal suppressive therapy is not usually effective for treating DIE. Interrupting the mechanical interactions between endometriotic fibroblasts and aberrant ECM may be a novel strategy for treatment of DIE. This study was supported in part by Karl Storz Endoscopy & GmbH (Tuttlingen, Germany). No competing interests are declared.

Abstract 3191: The role of rigidity in myeloid-tumor cell interactions in bone

Abstract Bone is one of the most common metastatic sites for breast cancer, where nearly 70% of patients that die from disease develop skeletal metastases. Skeletal metastases can cause hypercalcemia, fractures, and pain. With the use of 2D polyurethane films (PUR) that mimic tissue rigidity ranging from the basement membrane (10 MPa) to bone (2600 MPa), we have previously published that bone rigidity increases expression of Parathyroid hormone-related protein (PTHrP). This factor is important in tumor-induced bone disease in several cancer cell lines that metastasize to bone. Current in vivo studies implanting 3D scaffolds seeded with the bone metastatic breast cancer cells MDA-MB-231 GFP into athymic nude mice indicate that cells from the microenvironment infiltrate into the scaffolds, suggesting that these cells may play a role in tumor response to rigidity. Myeloid cells, which have been implicated in promoting tumor progression by altering the tumor microenvironment, are one of major infiltrating populations as determined by flow cytometry. Specifically CD11b+ myeloid cells made up 15% of overall cells within the scaffold, with the remaining population being mostly GFP+ tumor cells. Since few studies have examined the effect of rigidity on myeloid cells, we hypothesize that the physical rigidity of the bone promotes myeloid secretion of pro-tumorigenic factors that lead to an increase in tumor-expression of factors important in bone metastasis. In order to determine how rigidity influences myeloid cell differentiation and their effect on tumor progression, we seeded the macrophage/osteoclast cell line, RAW 264.7 cells on 2D PUR films. Differentiation of cells was monitored and conditioned media collected and used to treat MDA-MB-231 cells seeded on 2D PUR films. Surprisingly, we found that the conditioned media from RAW cells that were seeded onto the rigid films (bone-micking) decreased expression of PTHrP in tumor cells seeded on both rigid and soft films using RT-PCR. This effect was not seen from conditioned media from RAW cells seeded on soft films. Additionally, RAW cells seeded on soft films upregulate their expression of TGF-β, a factor important in regulating the expression of PTHrP, by 6-fold as compared to cells seeded on a rigid film. In contrast, PTHrP expression increases on rigid scaffolds in the absence of RAW conditioned media. Together these results suggest that myeloid cells sense changes in rigidity and alter their signaling profile accordingly to aid bone metastasis. Importantly, these studies highlight the need to perform studies that include more diverse cell populations, since the presence of myeloid cells or other cell types can alter gene expression within tumor cells. The use of 3D scaffolds will allow us to include multiple cell types and examine the interactions between tumor cells and cells in the bone microenvironment. Better understanding this interaction will help identify new therapeutic targets for the inhibition of tumor-induced bone disease. Citation Format: Denise Buenrostro, Ushashi Dadwal, Scott Guelcher, Julie Sterling. The role of rigidity in myeloid-tumor cell interactions in bone. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3191. doi:10.1158/1538-7445.AM2015-3191

Glucose sensing by carotid body glomus cells: potential implications in disease.

The carotid body (CB) is a key chemoreceptor organ in which glomus cells sense changes in blood O2, CO2, and pH levels. CB glomus cells have also been found to detect hypoglycemia in both non-primate mammals and humans. O2 and low-glucose responses share a common final pathway involving membrane depolarization, extracellular calcium influx, increase in cytosolic calcium concentration, and neurotransmitter secretion, which stimulates afferent sensory fibers to evoke sympathoadrenal activation. On the other hand, hypoxia and low glucose induce separate signal transduction pathways. Unlike O2 sensing, the response of the CB to low glucose is not altered by rotenone, with the low glucose-activated background cationic current unaffected by hypoxia. Responses of the CB to hypoglycemia and hypoxia can be potentiated by each other. The counter-regulatory response to hypoglycemia by the CB is essential for the brain, an organ that is particularly sensitive to low glucose. CB glucose sensing could be altered in diabetic patients, particularly those under insulin treatment, as well as in other medical conditions such as sleep apnea or obstructive pulmonary diseases, where chronic hypoxemia presents with plastic modifications in CB structure and function. The current review will focus on the following main aspects: (1) the CB as a low glucose sensor in both in vitro and in vivo models; (2) molecular and ionic mechanisms of low glucose sensing by glomus cells, (3) the interplay between low glucose and O2 sensing in CB, and (4) the role of CB low glucose sensing in the pathophysiology of cardiorespiratory and metabolic diseases, and how this may serve as a potential therapeutic target.

Threonine-4 of the budding yeast RNAP II CTD couples transcription with Htz1-mediated chromatin remodeling.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) consists of repeated YSPTSPS heptapeptides and connects transcription with cotranscriptional events. Threonine-4 (Thr4) of the CTD repeats has been shown to function in histone mRNA 3'-end processing in chicken cells and in transcriptional elongation in human cells. Here, we demonstrate that, in budding yeast, Thr4, although dispensable for growth in rich media, is essential in phosphate-depleted or galactose-containing media. Thr4 is required to maintain repression of phosphate-regulated (PHO) genes under normal growth conditions and for full induction of PHO5 and the galactose-induced GAL1 and GAL7 genes. We identify genetic links between Thr4 and the histone variant Htz1 and show that Thr4, as well as the Ino80 chromatin remodeler, is required for activation-associated eviction of Htz1 specifically from promoters of the Thr4-dependent genes. Our study uncovers a connection between transcription and chromatin remodeling linked by Thr4 of the CTD.

Low basolateral Na+ pump activity in macula densa cells may be necessary to generate tubuloglomerular feedback responses (892.32)

Macula densa (MD) cells sense changes in tubular fluid [Cl–] and this initiates a chain of events within the juxtaglomerular apparatus (i.e., the tubuloglomerular feedback (TGF) response) that regulates GFR. It is known that MD cells have much lower basolateral Na+/K+ATPase activity than neighboring cortical thick ascending limb (cTAL) cells. Also, an increase in luminal [NaCl] leads to the depolarization of the MD cell basolateral membrane, an event thought to be an essential step in the TGF response. To study the role of the Na+ pump in MD cells, we created a mathematical model that includes kinetic and thermodynamic models of the transport proteins and channels in MD cells. We examined the influence of luminal [NaCl] on basolateral membrane potential difference (PD). For comparison, similar computations were made with a model of a cTAL cell. When the luminal NaCl was varied from 20 to 150 mM, the basolateral membrane of the MD cell depolarized from –71 to –47 mV. For the cTAL cell, the basolateral membrane potential remained near –96 mV. These results suggest that the low expression of the basolateral Na+ pump may be appropriate in MD cells because, in these sensory cells, cytosolic [Na+] tracks luminal [Na+]. In a reabsorbing cTAL cell, the high basolateral Na+ pump expression stabilizes cytosolic [Na+] and basolateral PD.Grant Funding Source: Supported in part by MBRS‐RISE grant 2R25GM061151‐12 and by NIH‐NIGMS grant SC1GM084744.

Prolyl-4-hydroxylase domain 3 (PHD3) is a critical terminator for cell survival of macrophages under stress conditions

On a molecular level, cells sense changes in oxygen availability through the PHDs, which regulate the protein stability of the α-subunit of the transcription factor HIF. Especially, PHD3 has been additionally associated with apoptotic cell death. We hypothesized that PHD3 plays a role in cell-fate decisions in macrophages. Therefore, myeloid-specific PHD3(-/-) mice were created and analyzed. PHD3(-/-) BMDM showed no altered HIF-1α or HIF-2α stabilization or increased HIF target gene expression in normoxia or hypoxia. Macrophage M1 and M2 polarization was unchanged likewise. Compared with macrophages from WT littermates, PHD3(-/-) BMDM exhibited a significant reduction in TUNEL-positive cells after serum withdrawal or treatment with stauro and SNAP. Under the same conditions, PHD3(-/-) BMDM also showed less Annexin V staining, which is representative for membrane disruption, and indicated a reduced early apoptosis. In an unbiased transcriptome screen, we found that Angptl2 expression was reduced in PHD3(-/-) BMDM under stress conditions. Addition of rAngptl2 rescued the antiapoptotic phenotype, demonstrating that it is involved in the PHD3-mediated response toward apoptotic stimuli in macrophages.

Control of entry into meiosis and quiescence in budding yeast

How cells sense changes in environment and mount a response is a fundamental question in biology. Nutrition starvation in budding yeast has been an useful model to study this biological trait. When yeast cells are deprived of essential nutrients they can either enter a quiescent state (referred to as G0) or enter meiosis to form spores that can stay dormant for long periods of time. Precisely how environmental signals are sensed by yeast cells to trigger quiescence/meiosis is not known. Signals from multiple nutrient‐sensing signal transduction pathways converge on a serine‐threonine kinase called Rim15 which integrates the signals to orchestrate entry into quiescence or gametogenesis. In this we have identified a signalling pathway (REP) consisting of a serine‐threonine kinase Rim15, c‐AMP protein Igo1 and protein phosphatase PP2ACdc55 which regulates entry into both quiescence and meiosis. PP2ACdc55 negatively regulates entry into meiosis and G0. On nutrient starvation, Rim15 becomes active and phosphorylates endosulfines at Serine‐64 to convert it into an inhibitor of PP2ACdc55 thereby promoting entry into meiosis and G0. Remarkably a similar pathway controls entry into mitosis in mammalian cells.

Pyk2 regulates H+-ATPase-mediated proton secretion in the outer medullary collecting duct via an ERK1/2 signaling pathway

Acid-secreting intercalated cells respond to changes in systemic pH through regulation of apical H(+) transporters. Little is known about the mechanism by which these cells sense changes in extracellular pH (pH(o)). Pyk2 is a nonreceptor tyrosine kinase activated by autophosphorylation at Tyr402 by cell-specific stimuli, including decreased pH, and is involved in the regulation of MAPK signaling pathways and transporter activity. We examined whether the Pyk2 and MAPK signaling pathway mediates the response of transport proteins to decreased pH in outer medullary collecting duct cells. Immunoblot analysis of phosphorylated Pyk2 (Tyr402), ERK1/2 (Thr202/Tyr204), and p38 (Thr180/Tyr182) was used to assay protein activation. To examine specificity of kinase activation and its effects, we used Pyk2 small interfering RNA to knockdown Pyk2 expression levels, the Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP 1) to inhibit Pyk2 phosphorylation, and the MEK inhibitor U0126 to inhibit ERK1/2 phosphorylation. The pH-sensitive fluorescent probe 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) was used to assay H(+) transporter activity. The activity of H(+) transporters was measured as the rate of intracellular pH (pH(i)) recovery after an NH(4)Cl prepulse. We show that Pyk2 is endogenously expressed and activated by acid pH in mouse-derived outer medullary collecting duct (mOMCD1) cells. Incubation of mOMCD1 cells in acid media [extracellular pH (pH(o)) 6.7] increased the phosphorylation of Pyk2, ERK1/2, and p38. Reduction in pH(i) induced by an NH(4)Cl prepulse also increased the phosphorylation of Pyk2, ERK1/2, and p38. Consistent with our previous studies, we found that mOMCD1 cells exhibit H(+)-ATPase and H(+),K(+)-ATPase activity. Pyk2 inhibition by Pyk2 siRNA and PP 1 prevented Pyk2 phosphorylation as well as H(+)-ATPase-mediated recovery in mOMCD1 cells. In addition, ERK1/2 inhibition by U0126 prevented acid-induced ERK1/2 phosphorylation and H(+)-ATPase-mediated pH(i) recovery but not phosphorylation of p38. We conclude that Pyk2 and ERK1/2 are required for increasing H(+)-ATPase, but not H(+),K(+)-ATPase, activity at decreased pH(i) in mOMCD1 cells.