Autoregulatory Feedback Research Articles

Suppressive Role of ACVR1/ALK2 in Basal and TGFβ1-Induced Cell Migration in Pancreatic Ductal Adenocarcinoma Cells and Identification of a Self-Perpetuating Autoregulatory Loop Involving the Small GTPase RAC1b.

Pancreatic ductal adenocarcinoma (PDAC) cells are known for their high invasive/metastatic potential, which is regulated in part by the transforming growth factor β1 (TGFβ1). The involvement of at least two type I receptors, ALK5 and ALK2, that transmit downstream signals of the TGFβ via different Smad proteins, SMAD2/3 and SMAD1/5, respectively, poses the issue of their relative contribution in regulating cell motility. Real-time cell migration assays revealed that the selective inhibition of ALK2 by RNAi or dominant-negative interference with a kinase-dead mutant (ALK2-K233R) strongly enhanced the cells’ migratory activity in the absence or presence of TGFβ1 stimulation. Ectopic ALK2-K233R expression was associated with an increase in the protein levels of RAC1 and its alternatively spliced isoform, RAC1b, both of which are implicated in driving cell migration and invasion. Conversely, the RNAi-mediated knockdown or CRISPR/Cas9-mediated knockout of RAC1b resulted in the upregulation of the expression of ALK2, but not that of the related BMP type I receptors, ALK3 or ALK6, and elevated the phosphorylation of SMAD1/5. PDAC is a heterogeneous disease encompassing tumors with different histomorphological subtypes, ranging from epithelial/classical to extremely mesenchymal. Upon treatment of various established and primary PDAC cell lines representing these subtypes with the ALK2 inhibitor, LDN-193189, well-differentiated, epithelial cell lines responded with a much stronger increase in the basal and TGFβ1-dependent migratory activity than poorly differentiated, mesenchymal ones. These data show that (i) ALK2 inhibits migration by suppressing RAC1/RAC1b proteins, (ii) ALK2 and RAC1b act together in a self-perpetuating the autoregulatory negative feedback loop to mutually control their expression, and (iii) the ALK2 antimigratory function appears to be particularly crucial in protecting epithelial subtype cells from becoming invasive, both spontaneously and in a TGFβ-rich tumor microenvironment.

Circadian Regulation of Hormonal Timing and the Pathophysiology of Circadian Dysregulation.

Circadian rhythms are endogenously generated, daily patterns of behavior and physiology that are essential for optimal health and disease prevention. Disruptions to circadian timing are associated with a host of maladies, including metabolic disease and obesity, diabetes, heart disease, cancer, and mental health disturbances. The circadian timing system is hierarchically organized, with a master circadian clock located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus and subordinate clocks throughout the CNS and periphery. The SCN receives light information via a direct retinal pathway, synchronizing the master clock to environmental time. At the cellular level, circadian rhythms are ubiquitous, with rhythms generated by interlocking, autoregulatory transcription-translation feedback loops. At the level of the SCN, tight cellular coupling maintains rhythms even in the absence of environmental input. The SCN, in turn, communicates timing information via the autonomic nervous system and hormonal signaling. This signaling couples individual cellular oscillators at the tissue level in extra-SCN brain loci and the periphery and synchronizes subordinate clocks to external time. In the modern world, circadian disruption is widespread due to limited exposure to sunlight during the day, exposure to artificial light at night, and widespread use of light-emitting electronic devices, likely contributing to an increase in the prevalence, and the progression, of a host of disease states. The present overview focuses on the circadian control of endocrine secretions, the significance of rhythms within key endocrine axes for typical, homeostatic functioning, and implications for health and disease when dysregulated. © 2022 American Physiological Society. Compr Physiol 12: 1-30, 2022.

Patterns and Crucial Regulation of Alternative Splicing During Early Development in Zebrafish

Many vertebrate genes generate multiple transcript variants that may encode functionally distinct protein isoforms, but the transcriptomes of various developmental stages are poorly defined. Identifying the transcriptome and its regulation during the normal developmental process is the key to deciphering the developmental stage-specific functions of genes. Here we presented a systematic assessment of the temporal alternative splicing (AS) events during the critical development stages to capture the dynamic gene expression changes and AS in zebrafish. An unexpected transcriptome complexity generated by AS was observed during zebrafish development. The patterns of AS events varied substantially among developmental stages despite the similarities in the total proportion of AS genes. We further found that AS afforded substantial functional diversification of genes through the generation of stage-specific AS events from broadly protein-coding genes as an essential developmental regulatory mechanism. Skipped exon (SE) showed the strongest signals among developmental AS (devAS), suggesting that devAS events generated by SE may be necessary for the normal development of zebrafish. Most developmental genes regulated by AS mechanisms were not modulated in terms of their overall expression levels, indicating that AS shaped the transcriptome independently from transcriptional regulation during development. 128-cell stage was a critical stage for gene transcription during embryonic development. Splicing factors as an essential developmental regulator underwent AS in the potential autoregulatory feedback loop and expressed multiple isoforms. Thus, zebrafish development was shaped by an interplay of programs controlling gene expression levels and AS. Overall, we provided a global view of developmental patterns of AS during zebrafish development and revealed that AS transitions were the crucial regulatory component of zebrafish embryonic development.

Negative autoregulation controls size scaling in confined gene expression reactions

Gene expression via transcription-translation is the most fundamental reaction to sustain biological systems, and complex reactions occur in a small compartment of living cells. There is increasing evidence that physical effects, such as molecular crowding or excluded volume effects of transcriptional-translational machinery, affect the yield of reaction products. On the other hand, transcriptional feedback that controls gene expression during mRNA synthesis is also a vital mechanism that regulates protein synthesis in cells. However, the excluded volume effect of spatial constraints on feedback regulation is not well understood. Here, we study the confinement effect on transcriptional autoregulatory feedbacks of gene expression reactions using a theoretical model. The excluded volume effects between molecules and the membrane interface suppress the gene expression in a small cell-sized compartment. We find that negative feedback regulation at the transcription step mitigates this size-induced gene repression and alters the scaling relation of gene expression level on compartment volume, approaching the regular scaling relation without the steric effect. This recovery of regular size-scaling of gene expression does not appear in positive feedback regulation, suggesting that negative autoregulatory feedback is crucial for maintaining reaction products constant regardless of compartment size in heterogeneous cell populations.

Blimp-1 molds the epigenetic architecture of IL-21-mediated autoimmune diseases through an autoregulatory circuit.

Positive regulatory domain 1 (PRDM1) encodes B lymphocyte–induced maturation protein 1 (BLIMP1), also known as a master regulator of T cell homeostasis. We observed a negative relationship between Blimp-1 and IL-21 based on our previous data that Blimp-1 overexpression in T cells suppresses autoimmune diabetes while Blimp-1–deficient T cells contribute to colitis in NOD mice. Reanalysis of published data sets also revealed an inverse correlation between PRDM1 and IL21 in Crohn’s disease. Here, we illustrate that Blimp-1 repressed IL-21 by reducing chromatin accessibility and evicting an IL-21 activator, c-Maf, from the Il21 promoter. Moreover, Blimp-1 overexpression–mediated reduction in permissive chromatin structures at the Il21 promoter could override IL-21–accelerated autoimmune diabetogenesis in small ubiquitin-like modifier–defective c-Maf–transgenic mice. An autoregulatory feedback loop to harness IL-21 expression was unveiled by the evidence that IL-21 addition induced time-dependent Blimp-1 expression and subsequently enriched its binding to the Il21 promoter to suppress IL-21 overproduction. Furthermore, intervention of this feedback loop by IL-21 blockade, with IL-21R.Fc administration or IL-21 receptor deletion, attenuated Blimp-1 deficiency–mediated colitis and reinforced the circuit between Blimp-1 and IL-21 in the regulation of autoimmunity. We highlight the translation of Blimp-1–based epigenetic and transcriptomic profiles applicable to a personalized medicine approach in autoimmune diseases.

Circadian molecular clock disruption in chronic pulmonary diseases.

The circadian clock is the biochemical oscillator with a near 24-h period that is responsible for generating the circadian rhythms in peripheral organs including the lung. Mounting evidence suggests that circadian clock disruption during chronic lung diseases plays an essential role in augmented oxidative stress, inflammatory response, metabolic imbalances, hypoxia/hyperoxia, mucus secretion, dysregulated autophagy, and alters pulmonary function. Here, we review circadian clock disruption and discuss candidate clock genes that are altered at the transcriptional or translational level in chronic pulmonary diseases. This review aims to provide the current knowledge and understanding of the circadian molecular clock disruption in chronic pulmonary diseases which will further advance the development of novel clock-based therapeutics in the future.

Phosphorylation regulates E3 ligase activity of Mdm2

The Mdm2 oncogene is associated with the negative regulation of the tumor suppressor p53 in an autoregulatory feedback loop in response to DNA damage. It has been assumed that Mdm2 functions in this pathway as an E3 ubiquitin ligase and is Mdm2’s principal function under all conditions. However, the E3 ligase activity can function either as ubiquitin or nedd8 E3 ligase. This activity is dependent on specific tyrosine kinases that are activated in response to DNA damage signaling or growth factor signaling pathways. The E3 ligase activity can have a distinct effect on the substrate by either stabilizing or signaling destruction. In the context of growth signaling pathways, we show that Src phosphorylation of Mdm2 recruits Ubc12 (nedd8 E2 enzyme). This results in the neddylation of the tumor suppressors pVHL and p53. The neddylation of pVHL prevents the association with p53, thereby attenuating the induction of anti‐angiogenic genes. The phosphorylation of Mdm2 by Src is in the Zinc domain, and we found that this domain is a key regulatory domain for the recruitment of E2 enzymes. These findings suggest that the role of Mdm2 is more complex than just functioning as an E3 ubiquitin ligase to signal for the destruction of p53.

Molecular expression of clock genes in central and peripheral tissues of white-rumped munia (Lonchura striata)

ABSTRACT To synchronize with the fluctuating environment, organisms have evolved an endogenous time tracking mechanism referred to as the biological clock(s). This clock machinery has been identified in almost all cells of vertebrates and categorized as central and peripheral clocks. In birds, three independent circadian clocks have been identified in the hypothalamus, the pineal and the retina which interact and generate circadian time at a functional level. However, there is a limited knowledge of molecular clockwork and integration between central and peripheral clocks in birds. Therefore, we studied the daily expression of clock genes (Bmal1, Clock, Per2, Cry1, Npas2, Rev-Erbα, E4bp4, Pparα, Hlf and Tef) in three central circadian clocks (hypothalamus, pineal and retina), other brain areas (cerebellum, optic tectum and telencephalon) and in the peripheral tissues (liver, intestine, muscle and blood) of white-rumped munia. Adult birds were exposed to equinox photoperiod (12 L:12D) for 2 weeks and were then sampled (N = 5 per time point) at six-time points (ZT1, ZT5, ZT9, ZT13, ZT17 and ZT21). Daily expressions of clock genes were studied using qPCR. We observed daily variations and tissue-specific expression patterns for clock genes. These results are consistent with the autoregulatory circadian feedback loop proposed for the mammalian system and thus suggest a conserved tissue-level circadian time generation in white-rumped munia.

Physiological tissue-specific and age-related reduction of mouse TDP-43 levels is regulated by epigenetic modifications.

ABSTRACTThe cellular level of TDP-43 (also known as TARDBP) is tightly regulated; increases or decreases in TDP-43 have deleterious effects in cells. The predominant mechanism responsible for the regulation of the level of TDP-43 is an autoregulatory negative feedback loop. In this study, we identified an in vivo cause-effect relationship between Tardbp gene promoter methylation and specific histone modification and the TDP-43 level in tissues of mice at two different ages. Furthermore, epigenetic control was observed in mouse and human cultured cell lines. In amyotrophic lateral sclerosis, the formation of TDP-43-containing brain inclusions removes functional protein from the system. This phenomenon is continuous but compensated by newly synthesized protein. The balance between sequestration and new synthesis might become critical with ageing, if accompanied by an epigenetic modification-regulated decrease in newly synthesized TDP-43. Sequestration by aggregates would then decrease the amount of functional TDP-43 to a level lower than those needed by the cell and thereby trigger the onset of symptoms.

Functions of animal microRNA-encoded peptides: the race is on!

Short open reading frame (sORF)-encoded peptides (SEPs) recently emerged as new key players in biology. Pioneering work first established that sORFs encoded by long non-coding RNAs (lncRNAs) are efficiently translated and produce functional peptides. In plants, primary transcripts of microRNAs (pri-miRNAs) also produce sORF-encoded peptides called miPEPs, which are involved in specific transcriptional autoregulatory feedback loops (Lauressergues etal, 2015). To what extend are such mechanisms conserved in other species, especially in animals? In this issue of EMBO reports, Zhou etal show that pri-miR-31 encodes a miPEP promoting Treg differentiation and downregulating pri-miR-31 expression (Zhou etal, 2022).

"Reframing" dopamine signaling at the intersection of glial networks in the aged Parkinsonian brain as innate Nrf2/Wnt driver: Therapeutical implications.

Dopamine (DA) signaling via G protein‐coupled receptors is a multifunctional neurotransmitter and neuroendocrine–immune modulator. The DA nigrostriatal pathway, which controls the motor coordination, progressively degenerates in Parkinson's disease (PD), a most common neurodegenerative disorder (ND) characterized by a selective, age‐dependent loss of substantia nigra pars compacta (SNpc) neurons, where DA itself is a primary source of oxidative stress and mitochondrial impairment, intersecting astrocyte and microglial inflammatory networks. Importantly, glia acts as a preferential neuroendocrine–immune DA target, in turn, counter‐modulating inflammatory processes. With a major focus on DA intersection within the astrocyte–microglial inflammatory network in PD vulnerability, we herein first summarize the characteristics of DA signaling systems, the propensity of DA neurons to oxidative stress, and glial inflammatory triggers dictating the vulnerability to PD. Reciprocally, DA modulation of astrocytes and microglial reactivity, coupled to the synergic impact of gene–environment interactions, then constitute a further level of control regulating midbrain DA neuron (mDAn) survival/death. Not surprisingly, within this circuitry, DA converges to modulate nuclear factor erythroid 2‐like 2 (Nrf2), the master regulator of cellular defense against oxidative stress and inflammation, and Wingless (Wnt)/β‐catenin signaling, a key pathway for mDAn neurogenesis, neuroprotection, and immunomodulation, adding to the already complex “signaling puzzle,” a novel actor in mDAn–glial regulatory machinery. Here, we propose an autoregulatory feedback system allowing DA to act as an endogenous Nrf2/Wnt innate modulator and trace the importance of DA receptor agonists applied to the clinic as immune modifiers.

Circadian clock in plants: Linking timing to fitness.

Endogenous circadian clock integrates cyclic signals of environment and daily and seasonal behaviors of organisms to achieve spatiotemporal synchronization, which greatly improves genetic diversity and fitness of species. This review addresses recent studies on the plant circadian system in the field of chronobiology, covering topics on molecular mechanisms, internal and external Zeitgebers, and hierarchical regulation of physiological outputs. The architecture of the circadian clock involves the autoregulatory transcriptional feedback loops, post-translational modifications of core oscillators, and epigenetic modifications of DNA and histones. Here, light, temperature, humidity, and internal elemental nutrients are summarized to illustrate the sensitivity of the circadian clock to timing cues. In addition, the circadian clock runs cell-autonomously, driving independent circadian rhythms in various tissues. The core oscillators responds to each other with biochemical factors including calcium ions, mineral nutrients, photosynthetic products, and hormones. We describe clock components sequentially expressed during a 24-h day that regulate rhythmic growth, aging, immune response, and resistance to biotic and abiotic stresses. Notably, more data have suggested the circadian clock links chrono-culture to key agronomic traits in crops.

P2Y1 purinergic receptor identified as a diabetes target in a small-molecule screen to reverse circadian β-cell failure.

The mammalian circadian clock drives daily oscillations in physiology and behavior through an autoregulatory transcription feedback loop present in central and peripheral cells. Ablation of the core clock within the endocrine pancreas of adult animals impairs the transcription and splicing of genes involved in hormone exocytosis and causes hypoinsulinemic diabetes. Here, we developed a genetically sensitized small-molecule screen to identify druggable proteins and mechanistic pathways involved in circadian β-cell failure. Our approach was to generate β-cells expressing a nanoluciferase reporter within the proinsulin polypeptide to screen 2640 pharmacologically active compounds and identify insulinotropic molecules that bypass the secretory defect in CRISPR-Cas9-targeted clock mutant β-cells. We validated hit compounds in primary mouse islets and identified known modulators of ligand-gated ion channels and G-protein-coupled receptors, including the antihelmintic ivermectin. Single-cell electrophysiology in circadian mutant mouse and human cadaveric islets revealed ivermectin as a glucose-dependent secretagogue. Genetic, genomic, and pharmacological analyses established the P2Y1 receptor as a clock-controlled mediator of the insulinotropic activity of ivermectin. These findings identify the P2Y1 purinergic receptor as a diabetes target based upon a genetically sensitized phenotypic screen.

Activation and negative feedback regulation of SlHY5 transcription by the SlBBX20/21-SlHY5 transcription factor module in UV-B signaling.

In tomato (Solanum lycopersicum) and other plants, the photoreceptor UV-RESISTANCE LOCUS 8 regulates plant UV-B photomorphogenesis by modulating the transcription of many genes, the majority of which depends on the transcription factor ELONGATED HYPOCOTYL 5 (HY5). HY5 transcription is induced and then rapidly attenuated by UV-B. However, neither the transcription factors that activate HY5 transcription nor the mechanism for its attenuation during UV-B signaling is known. Here, we report that the tomato B-BOX (BBX) transcription factors SlBBX20 and SlBBX21 interact with SlHY5 and bind to the SlHY5 promoter to activate its transcription. UV-B-induced SlHY5 expression and SlHY5-controlled UV-B responses are normal in slbbx20 and slbbx21 single mutants, but strongly compromised in the slbbx20 slbbx21 double mutant. Surprisingly, UV-B responses are also compromised in lines overexpressing SlBBX20 or SlBBX21. Both SlHY5 and SlBBX20 bind to G-box1 in the SlHY5 promoter. SlHY5 outcompetes SlBBX20 for binding to the SlHY5 promoter in vitro, and inhibits the association of SlBBX20 with the SlHY5 promoter in vivo. Overexpressing 35S:SlHY5-FLAG in the WT background inhibits UV-B-induced endogenous SlHY5 expression. Together, our results reveal the critical role of the SlBBX20/21-SlHY5 module in activating the expression of SlHY5, the gene product of which inhibits its own gene transcription under UV-B, forming an autoregulatory negative feedback loop that balances SlHY5 transcription in plants.

Parameterising Translational Feedback Models of Autoregulatory RNA-Binding Proteins in Saccharomyces cerevisiae.

Post-transcriptional gene regulation is driven by RNA-binding proteins (RBPs). Recent global approaches suggest widespread autoregulation of RBPs through binding to their own mRNA; however, little is known about the regulatory impact and quantitative models remain elusive. By integration of several independent kinetic parameters and abundance data, we modelled autoregulatory feedback loops for six canonical and non-canonical RBPs from the yeast Saccharomyces cerevisiae, namely Hrb1p, Hek2/Khd1p, Ski2p, Npl3p, Pfk2p, and Map1p. By numerically solving ordinary differential equations, we compared non-feedback models with models that considered the RPBs as post-transcriptional activators/repressors of their own expression. While our results highlight a substantial gap between predicted protein output and experimentally determined protein abundances applying a no-feedback model, addition of positive feedback loops are surprisingly versatile and can improve predictions towards experimentally determined protein levels, whereas negative feedbacks are particularly sensitive to cooperativity. Our data suggests that introduction of feedback loops supported by real data can improve models of post-transcriptional gene expression.

Engagement of the CXCL12-CXCR4 Axis in the Interaction of Endothelial Progenitor Cell and Smooth Muscle Cell to Promote Phenotype Control and Guard Vascular Homeostasis.

Endothelial progenitor cells (EPCs) are involved in vascular repair and modulate properties of smooth muscle cells (SMCs) relevant for their contribution to neointima formation following injury. Considering the relevant role of the CXCL12–CXCR4 axis in vascular homeostasis and the potential of EPCs and SMCs to release CXCL12 and express CXCR4, we analyzed the engagement of the CXCL12–CXCR4 axis in various modes of EPC–SMC interaction relevant for injury- and lipid-induced atherosclerosis. We now demonstrate that the expression and release of CXCL12 is synergistically increased in a CXCR4-dependent mechanism following EPC–SMC interaction during co-cultivation or in response to recombinant CXCL12, thus establishing an amplifying feedback loop Additionally, mechanical injury of SMCs induces increased release of CXCL12, resulting in enhanced CXCR4-dependent recruitment of EPCs to SMCs. The CXCL12–CXCR4 axis is crucially engaged in the EPC-triggered augmentation of SMC migration and the attenuation of SMC apoptosis but not in the EPC-mediated increase in SMC proliferation. Compared to EPCs alone, the alliance of EPC–SMC is superior in promoting the CXCR4-dependent proliferation and migration of endothelial cells. When direct cell–cell contact is established, EPCs protect the contractile phenotype of SMCs via CXCL12–CXCR4 and reverse cholesterol-induced transdifferentiation toward a synthetic, macrophage-like phenotype. In conclusion we show that the interaction of EPCs and SMCs unleashes a CXCL12–CXCR4-based autoregulatory feedback loop promoting regenerative processes and mediating SMC phenotype control to potentially guard vascular homeostasis.

Modeling noise propagation in time-delayed auto-inhibitory genetic circuits

The abundance of specific protein molecules in genetically identical cell populations exposed to the same external environment can show remarkable cell-to-cell variations as biochemical reactions are inherently stochastic and occur with low numbers of molecular copies. Such variations in gene products are commonly known as gene expression noise. One of the mechanisms for cells to reduce such noise is auto-regulatory negative feedback (auto-inhibition), commonly found across organisms. This auto-inhibition is subjected to unavoidable time-delays associated with transcriptional and translational processes. Sufficient time-delays and strong auto-inhibition can generate sustained oscillations in gene products, which is a common mechanism for precise timekeeping in many biomolecular clocks. While the importance of time-delays in the generation of oscillations is well appreciated, its role in stochastic dynamics is not well understood in the absence of sustained oscillations. Here, we investigate the interplay between the feedback strength and the time-delay to study the noise propagation in the non-oscillatory regime using linear stability analysis, the linear noise approximation, and stochastic simulations. From a simple auto-regulatory model with one protein species (no delay), we systematically introduce one-step and two-step time-delays by incorporating intermediate dynamics with additional second and third species, respectively. Interestingly, the negative feedback in the presence of time-delay can show counterintuitive noise behavior to our common perception about its role as a noise buffer.

Type I CRISPR-Cas provides robust immunity but incomplete attenuation of phage-induced cellular stress.

During infection, phages manipulate bacteria to redirect metabolism towards viral proliferation. To counteract phages, some bacteria employ CRISPR-Cas systems that provide adaptive immunity. While CRISPR-Cas mechanisms have been studied extensively, their effects on both the phage and the host during phage infection remains poorly understood. Here, we analysed the infection of Serratia by a siphovirus (JS26) and the transcriptomic response with, or without type I-E or I-F CRISPR-Cas immunity. In non-immune Serratia, phage infection altered bacterial metabolism by upregulating anaerobic respiration and amino acid biosynthesis genes, while flagella production was suppressed. Furthermore, phage proliferation required a late-expressed viral Cas4 homologue, which did not influence CRISPR adaptation. While type I-E and I-F immunity provided robust defence against phage infection, phage development still impacted the bacterial host. Moreover, DNA repair and SOS response pathways were upregulated during type I immunity. We also discovered that the type I-F system is controlled by a positive autoregulatory feedback loop that is activated upon phage targeting during type I-F immunity, leading to a controlled anti-phage response. Overall, our results provide new insight into phage-host dynamics and the impact of CRISPR immunity within the infected cell.

Acidic environments trigger intracellular H+-sensing FAK proteins to re-balance sarcolemmal acid-base transporters and auto-regulate cardiomyocyte pH.

In cardiomyocytes, acute disturbances to intracellular pH (pHi) are promptly corrected by a system of finely tuned sarcolemmal acid-base transporters. However, these fluxes become thermodynamically re-balanced in acidic environments, which inadvertently causes their set-point pHi to fall outside the physiological range. It is unclear whether an adaptive mechanism exists to correct this thermodynamic challenge, and return pHi to normal. Following left ventricle cryo-damage, a diffuse pattern of low extracellular pH (pHe) was detected by acid-sensing pHLIP. Despite this, pHi measured in the beating heart (13C NMR) was normal. Myocytes had adapted to their acidic environment by reducing Cl-/HCO3- exchange (CBE)-dependent acid-loading and increasing Na+/H+ exchange (NHE1)-dependent acid-extrusion, as measured by fluorescence (cSNARF1). The outcome of this adaptation on pHi is revealed as a cytoplasmic alkalinization when cells are superfused at physiological pHe. Conversely, mice given oral bicarbonate (to improve systemic buffering) had reduced myocardial NHE1 expression, consistent with a needs-dependent expression of pHi-regulatory transporters. The response to sustained acidity could be replicated in vitro using neonatal ventricular myocytes incubated at low pHe for 48 h. The adaptive increase in NHE1 and decrease in CBE activities was linked to Slc9a1 (NHE1) up-regulation and Slc4a2 (AE2) down-regulation. This response was triggered by intracellular H+ ions because it persisted in the absence of CO2/HCO3- and became ablated when acidic incubation media had lower chloride, a solution manoeuvre that reduces the extent of pHi-decrease. Pharmacological inhibition of FAK-family non-receptor kinases, previously characterized as pH-sensors, ablated this pHi autoregulation. In support of a pHi-sensing role, FAK protein Pyk2 (auto)phosphorylation was reduced within minutes of exposure to acidity, ahead of adaptive changes to pHi control. Cardiomyocytes fine-tune the expression of pHi-regulators so that pHi is at least 7.0. This autoregulatory feedback mechanism defines physiological pHi and protects it during pHe vulnerabilities.

Circadian clock dysfunction of epithelial cells in pulmonary diseases.

Highly-differentiated pulmonary epithelial cells are essential for maintaining lung homeostasis by exerting various physiological functions, which are regulated by circadian clock consisted of an autoregulatory feedback loop of clock genes, including Brain-Muscle Aryl-hydrocarbon Receptor Nuclear Translocator-Like 1 (BMAL1) and Nuclear Heme Receptor Reverse Erythroblastosis Virus α (REV-ERB-α). The circadian clock dysfunction of epithelial cells has been increasingly associated with the pulmonary diseases: BMAL1 and REV-ERB-α regulates inflammatory response of club cells induced by lipopolysaccharide and cigarette smoke (CS) respectively; the clock disfunction in alveolar epithelial type2 cells (AEC-II) has been implicated in CS-induced airway inflammation and early-life hyperoxia-related susceptibility to influenza infection; the ciliary beat frequency of ciliated cells also shows circadian rhythms. Here, we review the current knowledge on the circadian regulation of different epithelial-cell subtypes, attempting to provide insights into how clock dysfunction contributes to pulmonary diseases, and explore possible pharmacological therapies and future directions for fundamental studies.