Analysis of the DNA-binding properties of TGF-ß-activated Smad complexes unveils a possible molecular basis for cellular context-dependent signaling.

Transforming growth factor-ß (TGF-ß) is a pleiotropic cytokine that modulates a wide variety of cellular responses by regulating target gene expression. It principally transmits signals via receptor-activated transcription factors Smad2 and Smad3, which form trimeric complexes with Smad4 upon activation and regulate gene expression by binding to genomic DNA. Here, we examined the mechanisms by which TGF-ß regulates the transcription of target genes in a cell context-dependent manner by screening a double-stranded DNA oligonucleotide library for DNA sequences bound to endogenous activated Smad complexes. Screening was performed by cyclic amplification of selected targets (CASTing) using an anti-Smad2/3 antibody and nuclear extracts isolated from three cell lines (A549, HepG2, and HaCaT) stimulated with TGF-ß. The preference of the activated Smad complexes for conventional Smad-binding motifs such as Smad-binding element (SBE) and CAGA motifs was different in HepG2 than in the other two cell lines, which may indicate the distinct composition of the activated Smad complexes. Several transcription factor-binding motifs other than SBE or CAGA, including the Fos/Jun-binding motifs, were detected in the enriched sequences. Reporter assays using sequences containing these transcription factor-binding motifs together with Smad-binding motifs indicated that some of the motifs may be involved in cell type-dependent transcriptional activation by TGF-ß. The results suggest that the CASTing method is useful for elucidating the molecular basis of context-dependent Smad signaling.

Medication Optimization Protocol Efficacy for Geriatric Inpatients: A Randomized Clinical Trial.

Importance: There is currently no consensus on clinically effective interventions for polypharmacy among older inpatients. Objective: To evaluate the effect of multidisciplinary team-based medication optimization on survival, unscheduled hospital visits, and rehospitalization in older inpatients with polypharmacy. Design, Setting, and Participants: This open-label randomized clinical trial was conducted at 8 internal medicine inpatient wards within a community hospital in Japan. Participants included medical inpatients 65 years or older who were receiving 5 or more regular medications. Enrollment took place between May 21, 2019, and March 14, 2022. Statistical analysis was performed from September 2023 to May 2024. Intervention: The participants were randomly assigned to receive either an intervention for medication optimization or usual care including medication reconciliation. The intervention consisted of a medication review using the STOPP (Screening Tool of Older Persons' Prescriptions)/START (Screening Tool to Alert to Right Treatment) criteria, followed by a medication optimization proposal for participants and their attending physicians developed by a multidisciplinary team. On discharge, the medication optimization summary was sent to patients' primary care physicians and community pharmacists. Main Outcomes and Measures: The primary outcome was a composite of death, unscheduled hospital visits, and rehospitalization within 12 months. Secondary outcomes included the number of prescribed medications, falls, and adverse events. Results: Between May 21, 2019, and March 14, 2022, 442 participants (mean [SD] age, 81.8 [7.1] years; 223 [50.5%] women) were randomly assigned to the intervention (n = 215) and usual care (n = 227). The intervention group had a significantly lower percentage of patients with 1 or more potentially inappropriate medications than the usual care group at discharge (26.2% vs 33.0%; adjusted odds ratio [OR], 0.56 [95% CI, 0.33-0.94]; P = .03), at 6 months (27.7% vs 37.5%; adjusted OR, 0.50 [95% CI, 0.29-0.86]; P = .01), and at 12 months (26.7% vs 37.4%; adjusted OR, 0.45 [95% CI, 0.25-0.80]; P = .007). The primary composite outcome occurred in 106 participants (49.3%) in the intervention group and 117 (51.5%) in the usual care group (stratified hazard ratio, 0.98 [95% CI, 0.75-1.27]). Adverse events were similar between each group (123 [57.2%] in the intervention group and 135 [59.5%] in the usual care group). Conclusions and Relevance: In this randomized clinical trial of older inpatients with polypharmacy, the multidisciplinary deprescribing intervention did not reduce death, unscheduled hospital visits, or rehospitalization within 12 months. The intervention was effective in reducing the number of medications with no significant adverse effects on clinical outcomes, even among older inpatients with polypharmacy. Trial Registration: UMIN Clinical Trials Registry: UMIN000035265.

Receptor-activated transcription factors and beyond: multiple modes of Smad2/3-dependent transmission of TGF-ß signaling.

Transforming growth factor ß (TGF-ß) is a pleiotropic cytokine that is widely distributed throughout the body. Its receptor proteins, TGF-ß type I and type II receptors, are also ubiquitously expressed. Therefore, the regulation of various signaling outputs in a context-dependent manner is a critical issue in this field. Smad proteins were originally identified as signal-activated transcription factors similar to signal transducer and activator of transcription proteins. Smads are activated by serine phosphorylation mediated by intrinsic receptor dual specificity kinases of the TGF-ß family, indicating that Smads are receptor-restricted effector molecules downstream of ligands of the TGF-ß family. Smad proteins have other functions in addition to transcriptional regulation, including post-transcriptional regulation of micro-RNA processing, pre-mRNA splicing, and m6A methylation. Recent technical advances have identified a novel landscape of Smad-dependent signal transduction, including regulation of mitochondrial function without involving regulation of gene expression. Therefore, Smad proteins are receptor-activated transcription factors and also act as intracellular signaling modulators with multiple modes of function. In this review, we discuss the role of Smad proteins as receptor-activated transcription factors and beyond. We also describe the functional differences between Smad2 and Smad3, two receptor-activated Smad proteins downstream of TGF-ß, activin, myostatin, growth and differentiation factor (GDF) 11, and Nodal.

Smad2Δexon3 and Smad3 have distinct properties in signal transmission leading to TGF-ß-induced cell motility.

In mammalian cells, Smad2 and Smad3, two receptor-regulated Smad proteins, play crucial roles in the signal transmission of transforming growth factor-ß (TGF-ß) and are involved in various cell regulatory processes, including epithelial-mesenchymal transition-associated cell responses, that is, cell morphological changes, E-cadherin downregulation, stress fiber formation, and cell motility enhancement. Smad2 contains an additional exon encoding 30 amino acid residues compared with Smad3, leading to distinct Smad2 and Smad3 functional properties. Intriguingly, Smad2 also has an alternatively spliced isoform termed Smad2Δexon3 (also known as Smad2ß) lacking the additional exon and behaving similarly to Smad3. However, Smad2Δexon3 and Smad3 signaling properties have not yet been compared in detail. In this study, we reveal that Smad2Δexon3 rescues multiple TGF-ß-induced in vitro cellular responses that would become defective upon SMAD3 KO but does not rescue cell motility enhancement. Using Smad2Δexon3/Smad3 chimeric proteins, we identified that residues Arg-104 and Asn-210 in Smad3, which are not conserved in Smad2Δexon3, are key for TGF-ß-enhanced cell motility. Moreover, we discovered that Smad2Δexon3 fails to rescue the enhanced cell motility as it does not mediate TGF-ß signals to downregulate transcription of ARHGAP24, a GTPase-activating protein that targets Rac1. This study reports for the first time distinct signaling properties of Smad2Δexon3 and Smad3.

Indole-derived compound SIS3 targets a subset of activated Smad complexes.

Smad2 and Smad3 are receptor-regulated Smad proteins that transmit signals from cytokines belonging to the transforming growth factor (TGF)-ß family, which are vital for adult tissue homeostasis. The overactivation of such proteins often engenders the development of pathological conditions. Smad3 reportedly mediates TGF-ß-induced fibrosis. Although various potential Smad3-specific inhibitors are being developed, their specificity and action mechanisms remain largely unknown. This study aimed to establish a biochemical platform to monitor Smad2- or Smad3-dependent TGF-ß signaling using SMAD2, SMAD3 and SMAD2/3 knockout cell lines alongside TGF-ß-dependent luciferase reporters and Smad mutant proteins. Using this platform, SIS3, an indole-derived compound widely used as a specific Smad3 inhibitor, was observed to preferentially suppress a subset of activated Smad complexes. However, its inhibition did not favor Smad3 signaling over Smad2 signaling. These findings indicate that SIS3 can be employed as a probe to examine the heterogeneous nature of Smad signaling that induces gene expression. However, its use as a Smad3-specific inhibitor should be avoided.

Ets family proteins regulate the EMT transcription factors Snail and ZEB in cancer cells.

The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during epithelial tumor progression. Snail and ZEB1/2 (ZEB1 and ZEB2), known as EMT transcription factors, are key regulators of this transition. ZEB1/2 are positively correlated with EMT phenotypes and the aggressiveness of cancers. On the contrary, Snail is also correlated with the aggressiveness of cancers, but is not correlated with the expression of EMT marker proteins. Snail is induced by transforming growth factor-ß (TGF-ß), a well-known inducer of EMT, in various cancer cells. Interestingly, Snail induction by TGF-ß is markedly enhanced by active Ras signals. Thus, cancer cells harboring an active Ras mutation exhibit a drastic induction of Snail by TGF-ß alone. Here, we found that members of the E26 transformation-specific (Ets) transcription factor family, Ets1 and Ets2, contribute to the upregulation of both Snail and ZEB1/2. Snail induction by TGF-ß and active Ras is dramatically inhibited using siRNAs against both Ets1 and Ets2 together, but not on their own; in addition, siRNAs against both Ets1 and Ets2 also downregulate the constitutive expression of Snail and ZEB1/2 in cancer cells. Examination of several alternatively spliced variants of Ets1 revealed that p54-Ets1, which includes exon VII, but not p42-Ets1, which excludes exon VII, regulates the expression of the EMT transcription factors, suggesting that Ets1 is a crucial molecule for regulating Snail and ZEB1/2, and thus cancer progression is mediated through post-translational modification of the exon VII domain.

Periplocin and cardiac glycosides suppress the unfolded protein response.

The unfolded protein response (UPR) controls protein homeostasis through transcriptional and translational regulation. However, dysregulated UPR signaling has been associated with the pathogenesis of many human diseases. Therefore, the compounds modulating UPR may provide molecular insights for these pathologies in the context of UPR. Here, we screened small-molecule compounds that suppress UPR, using a library of Myanmar wild plant extracts. The screening system to track X-box binding protein 1 (XBP1) splicing activity revealed that the ethanol extract of the Periploca calophylla stem inhibited the inositol-requiring enzyme 1 (IRE1)-XBP1 pathway. We isolated and identified periplocin as a potent inhibitor of the IRE1-XBP1 axis. Periplocin also suppressed other UPR axes, protein kinase R-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). Examining the structure-activity relationship of periplocin revealed that cardiac glycosides also inhibited UPR. Moreover, periplocin suppressed the constitutive activation of XBP1 and exerted cytotoxic effects in the human multiple myeloma cell lines, AMO1 and RPMI8226. These results reveal a novel suppressive effect of periplocin or the other cardiac glycosides on UPR regulation, suggesting that these compounds will contribute to our understanding of the pathological or physiological importance of UPR.

TGF-ß-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

Transforming growth factor-ß (TGF-ß) signaling promotes cancer progression. In particular, the epithelial-mesenchymal transition (EMT) induced by TGF-ß is considered crucial to the malignant phenotype of cancer cells. Here, we report that the EMT-associated cellular responses induced by TGF-ß are mediated by distinct signaling pathways that diverge at Smad3. By expressing chimeric Smad1/Smad3 proteins in SMAD3 knockout A549 cells, we found that the ß4 region in the Smad3 MH1 domain is essential for TGF-ß-induced cell motility, but is not essential for other EMT-associated responses including epithelial marker downregulation. TGF-ß was previously reported to enhance cell motility by activating Rac1 via phosphoinositide 3-kinase. Intriguingly, TGF-ß-dependent signaling mediated by Smad3's ß4 region causes the downregulation of multiple mRNAs that encode GTPase activating proteins that target Rac1 (ARHGAPs), thereby attenuating Rac1 inactivation. Therefore, two independent pathways downstream of TGF-ß type I receptor contribute cooperatively to sustained Rac1 activation, thereby leading to enhanced cell motility.

ZEB1 and oncogenic Ras constitute a regulatory switch for stimulus-dependent E-cadherin downregulation.

E-cadherin, an epithelial cell-specific cell adhesion molecule, has both promoting and suppressing effects on tumor invasion and metastasis. It is often downregulated during cancer progression through gene deletion/mutation, transcriptional repression, or epigenetic silencing. We describe a novel regulatory switch to induce stimulus-dependent downregulation of mRNA encoding E-cadherin (CDH1 mRNA) in KRAS-mutated cancer cells. The regulatory switch consists of ZEB1 and oncogenic K-Ras, does not target the promoter region of CDH1, and requires an external cue to temporally downregulate E-cadherin expression. Its repressive effect is maintained as long as the external stimulus continues and is attenuated with cessation of the stimulus. Contextual external cues that turn this regulatory switch on include activation of protein kinase C or fibroblast growth factor signaling. The mode of action is distinct from that of EPCAM repression by ZEB1, which does not require an external cue. Thus, KRAS-mutated cancer cells acquire a novel mode of regulating E-cadherin expression depending on ZEB1, which could contribute to phenotypic plasticity of cancer cells during malignant progression.

Structural basis for inhibitory effects of Smad7 on TGF-ß family signaling.

Smad6 and Smad7 are classified as inhibitory Smads (I-Smads). They are crucial in the fine-tuning of signals by cytokines of the transforming growth factor-ß (TGF-ß) family. They are negative feedback regulators and principally target the activated type I receptors as well as the activated Smad complexes, but with distinct specificities. Smad7 inhibits Smad signaling from all seven type I receptors of the TGF-ß family, whereas Smad6 preferentially inhibits Smad signaling from the bone morphogenetic protein (BMP) type I receptors, BMPR1A and BMPR1B. The target specificities are attributed to the C-terminal MH2 domain. Notably, Smad7 utilizes two alternative molecular surfaces for its inhibitory function against type I receptors. One is a basic groove composed of the first α-helix and the L3 loop, a structure that is shared with Smad6 and receptor-regulated Smads (R-Smads). The other is a three-finger-like structure (consisting of residues 331-361, 379-387, and the L3 loop) that is unique to Smad7. The underlying structural basis remains to be elucidated in detail. Here, we report the crystal structure of the MH2 domain of mouse Smad7 at 1.9 Å resolution. The three-finger-like structure is stabilized by a network of hydrogen bonds between residues 331-361 and 379-387, thus forming a molecular surface unique to Smad7. Furthermore, we discuss how Smad7 antagonizes the activated Smad complexes composed of R-Smad and Smad4, a common partner Smad.

Protocol of a randomised controlled trial on the efficacy of medication optimisation in elderly inpatients: medication optimisation protocol efficacy for geriatric inpatients (MPEG) trial.

INTRODUCTION: Whether medication optimisation improves clinical outcomes in elderly individuals remains unclear. The current study aims to evaluate the effect of multidisciplinary team-based medication optimisation on survival, rehospitalisation and unscheduled hospital visits in elderly patients. METHODS AND ANALYSIS: We report the protocol of a single-centre, open-label, randomised controlled trial. The enrolled subjects will be medical inpatients, aged 65 years or older, admitted to a community hospital and receiving five or more regular medications. The participants will be randomly assigned to receive either an intervention for medication optimisation or the usual care. The intervention will consist of a multidisciplinary team-based medication review, followed by a medication optimisation proposal based on the Screening Tool of Older Persons' potentially inappropriate Prescriptions/Screening Tool to Alert doctors to the Right Treatment criteria and an implicit medication optimisation protocol. Medication optimisation summaries will be sent to primary care physicians and community pharmacists on discharge. The primary outcome will be a composite of death, unscheduled hospital visits and rehospitalisation until 48 weeks after randomisation. Secondary outcomes will include each of the primary endpoints, the number of prescribed medications, quality of life score, level of long-term care required, drug-related adverse events, death during hospitalisation and falls. Participants will be followed up for 48 weeks with bimonthly telephone interviews to assess the primary and secondary outcomes. A log-rank test stratified by randomisation factors will be used to compare the incidence of the composite endpoint. The study was initiated in 2019 and a minimum of 500 patients will be enrolled. ETHICS AND DISSEMINATION: The study protocol has been approved by the Institutional Ethical Committee of St. Marianna University School of Medicine (No. 4129). The results of the current study will be submitted to a peer-reviewed journal. TRIAL REGISTRATION NUMBER: UMIN000035265.

A comparative analysis of Smad-responsive motifs identifies multiple regulatory inputs for TGF-ß transcriptional activation.

Smad proteins are transcriptional regulators activated by TGF-ß. They are known to bind to two distinct Smad-responsive motifs, namely the Smad-binding element (SBE) (5'-GTCTAGAC-3') and CAGA motifs (5'-AGCCAGACA-3' or 5'-TGTCTGGCT-3'). However, the mechanisms by which these motifs promote Smad activity are not fully elucidated. In this study, we performed DNA CASTing, binding assays, ChIP sequencing, and quantitative RT-PCR to dissect the details of Smad binding and function of the SBE and CAGA motifs. We observed a preference for Smad3 to bind CAGA motifs and Smad4 to bind SBE, and that either one SBE or a triple-CAGA motif forms a cis-acting functional half-unit for Smad-dependent transcription activation; combining two half-units allows efficient activation. Unexpectedly, the extent of Smad binding did not directly correlate with the abilities of Smad-binding sequences to induce gene expression. We found that Smad proteins are more tolerant of single bp mutations in the context of the CAGA motifs, with any mutation in the SBE disrupting function. CAGA and CAGA-like motifs but not SBE are widely distributed among stimulus-dependent Smad2/3-binding sites in normal murine mammary gland epithelial cells, and the number of CAGA and CAGA-like motifs correlates with fold-induction of target gene expression by TGF-ß. These data, demonstrating Smad responsiveness can be tuned by both sequence and number of repeats, provide a compelling explanation for why CAGA motifs are predominantly used for Smad-dependent transcription activation in vivo.

Kurarinone from Sophora Flavescens Roots Triggers ATF4 Activation and Cytostatic Effects Through PERK Phosphorylation.

In response to cellular stresses, activating transcriptional factor 4 (ATF4) regulates the expression of both stress-relieving genes and apoptosis-inducing genes, eliciting cell fate determination. Since pharmacological activation of ATF4 exerts potent anti-tumor effects, modulators of ATF4 activation may have potential in cancer therapy. We herein attempted to identify small molecules that activate ATF4. A cell-based screening to monitor TRB3 promoter activation was performed using crude drugs used in traditional Japanese Kampo medicine. We found that an extract from Sophora flavescens roots exhibited potent TRB3 promoter activation. The activity-guided fractionation revealed that kurarinone was identified as the active ingredient. Intriguingly, ATF4 activation in response to kurarinone required PKR-like endoplasmic reticulum kinase (PERK). Moreover, kurarinone induced the cyclin-dependent kinase inhibitor p21 as well as cytostasis in cancer cells. Importantly, the cytostatic effect of kurarinone was reduced by pharmacological inhibition of PERK. These results indicate that kurarinone triggers ATF4 activation through PERK and exerts cytostatic effects on cancer cells. Taken together, our results suggest that modulation of the PERK-ATF4 pathway with kurarinone has potential as a cancer treatment.

TRB1 negatively regulates gluconeogenesis by suppressing the transcriptional activity of FOXO1.

Tribbles related homolog 1 is the mammalian ortholog of Tribbles, which controls cell division and migration during development in Drosophila. TRB1 is a pseudokinase and functions as a scaffold protein. Recent findings suggest that TRB1 plays important roles in hepatic lipid metabolism and participates in insulin resistance. However, the underlying mechanisms have not yet been elucidated. Here, we demonstrate that TRB1 suppresses FOXO1 transcriptional activity to downregulate the expression of G6Pase and PEPCK, which encode gluconeogenic rate-limiting enzymes. TRB1 knockdown enhances FOXO1 binding to the gluconeogenic gene promoters. It also increases FOXO1 acetylation and recruits CBP to the binding sequence of FOXO1. These results suggest that TRB1 suppresses the expression of G6Pase and PEPCK by attenuating FOXO1 transcriptional activity and negatively regulates gluconeogenesis.

Anti-Tumorigenic Activity of Chrysin from Oroxylum indicum via Non-Genotoxic p53 Activation through the ATM-Chk2 Pathway.

The p53 tumor suppressor plays critical roles in cell cycle regulation and apoptotic cell death in response to various cellular stresses, thereby preventing cancer development. Therefore, the activation of p53 through small molecules is an attractive therapeutic strategy for the treatment of cancers retaining wild-type p53. We used a library of 700 Myanmar wild plant extracts to identify small molecules that induce p53 transcriptional activity. A cell-based screening method with a p53-responsive luciferase-reporter assay system revealed that an ethanol extract of Oroxylum indicum bark increased p53 transcriptional activity. Chrysin was isolated and identified as the active ingredient in the O. indicum bark extract. A treatment with chrysin increased p53 protein expression and the p53-mediated expression of downstream target genes, and decreased cell viability in MCF7 cells, but not in p53-knockdown MCF7 cells. We also found that chrysin activated the ATM-Chk2 pathway in the absence of DNA damage. Hence, the inactivation of the ATM-Chk2 pathway suppressed p53 activation induced by chrysin. These results suggest the potential of chrysin as an anti-cancer drug through the activation of p53 without DNA damage.

Smad3-STAT3 crosstalk in pathophysiological contexts.

Smad3 and STAT3 are intracellular molecules that transmit signals from plasma membrane receptors to the nucleus. Smad3 operates downstream of growth/differentiation factors that utilize activin receptor-like kinase (ALK)-4, 5, or 7, such as transforming growth factor-ß (TGF-ß), activin, and myostatin. STAT3 principally functions downstream of cytokines that exert their effects via gp130 and Janus family kinases, including interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and oncostatin M. Accumulating evidence indicates that Smad3 and STAT3 engage in crosstalk in a highly context-dependent fashion, cooperating in some conditions while acting antagonistically each other in others. Here, we review the crosstalk between Smad3 and STAT3 in various biological contexts, including early tumorigenesis, epithelial-mesenchymal transition, fibrosis, and T cell differentiation.

The CDK inhibitor p21 is a novel target gene of ATF4 and contributes to cell survival under ER stress.

Activating transcription factor 4 (ATF4) is well known for its role in the endoplasmic reticulum (ER) stress response. ATF4 also transcriptionally induces multiple effectors that determine cell fate depending on cellular context. In addition, ATF4 can communicate both pro-apoptotic and pro-survival signals. How ATF4 mediates its prosurvival roles, however, requires further investigation. Here, we report that the CDK inhibitor p21 is a novel target gene of ATF4. We identified two ATF4-responsive elements, one of which directly binds ATF4, within the first intron of the p21 gene. Importantly, overexpression of p21 enhances cell survival following ER stress induction, while p21 knockdown increases cell death. These results suggest that p21 induction plays a vital role in the cellular response to ER stress and indicate that p21 is a prosurvival effector of ATF4.

TGF-ß induces p53/Smads complex formation in the PAI-1 promoter to activate transcription.

Transforming growth factor ß (TGF-ß) signaling facilitates tumor development during the advanced stages of tumorigenesis, but induces cell-cycle arrest for tumor suppression during the early stages. However, the mechanism of functional switching of TGF-ß is still unknown, and it is unclear whether inhibition of TGF-ß signaling results amelioration or exacerbation of cancers. Here we show that the tumor suppressor p53 cooperates with Smad proteins, which are TGF-ß signal transducers, to selectively activate plasminogen activator inhibitor type-1 (PAI-1) transcription. p53 forms a complex with Smad2/3 in the PAI-1 promoter to recruit histone acetyltransferase CREB-binding protein (CBP) and enhance histone H3 acetylation, resulting in transcriptional activation of the PAI-1 gene. Importantly, p53 is required for TGF-ß-induced cytostasis and PAI-1 is involved in the cytostatic activity of TGF-ß in several cell lines. Our results suggest that p53 enhances TGF-ß-induced cytostatic effects by activating PAI-1 transcription, and the functional switching of TGF-ß is partially caused by p53 mutation or p53 inactivation during cancer progression. It is expected that these findings will contribute to optimization of TGF-ß-targeting therapies for cancer.

Blunted blood pressure response during hyperpnoea in endurance runners.

UNLABELLED: The purpose of this study was to elucidate the cardiovascular response during hyperpnoea in endurance-trained runners compared to sedentary controls. Twelve runners and ten sedentary individuals participated in this study. A maximal respiratory endurance test (MRET) was performed as follows: target minute ventilation was initially set at 30% of maximal voluntary ventilation (MVV12) and was increased by 10% MVV12 every 3min. The test was terminated when the subject could no longer maintain the target ventilation. Heart rate and mean arterial blood pressure (MBP) were continuously measured. Respiratory endurance time during the MRET was longer in the runners than the controls. The change in MBP during the MRET was lower in the runners compared to the sedentary controls (runners: 100.2±2.4mmHg vs. CONTROLS: 109.1±3.0mmHg at 6min of hyperpnoea). Therefore, the blood pressure response during hyperpnoea is blunted in endurance runners, suggesting that whole-body endurance exercise training attenuates the respiratory muscle-induced metaboreflex.