Consistently, our clinical observational analyses confirmed that higher blood UA levels, as well as the SLC2A9/GLUT9 rs11722228 T/T genotype, were associated with higher lung function in elderly human females. Together, our findings provide the first unique evidence that higher blood UA is a protective factor against the pathological decline of lung function in female mice, and possibly against aging-associated physiological decline in human females.The oxidant/antioxidant imbalance plays a pivotal role in the lung. Uric acid (UA), an endogenous antioxidant, is highly present in lung tissue, however, its impact on lung function under pathophysiological conditions remains unknown. In this work, pharmacological and genetic inhibition of UA metabolism in experimental mouse models of acute and chronic obstructive pulmonary disease (COPD) revealed that increased plasma UA levels improved emphysematous phenotype and lung dysfunction in accordance with reduced oxidative stress specifically in female but not in male mice, despite no impact of plasma UA induction on the pulmonary phenotypes in nondiseased mice. In vitro experiments determined that UA significantly suppressed hydrogen peroxide (H Respiratory dysfunction affects quality of life and facilitates the development of noncommunicable chronic lung diseases such as chronic obstructive pulmonary disease (COPD), a condition characterized by symptoms such as increased average airspace size, decreased elasticity, and decreased forced expiratory volume in one second FEV1)/forced vital capacity (FVC) /forced v,3. ConsiAmong the endogenous antioxidants in epithelial lining fluid (ELF), uric acid (UA) exists in higher concentrations  and has 2O2)-induced oxidative stress only in the absence of estrogen, implying that the benefit of higher UA is limited to the female airway in postmenopausal conditions. We also conducted a cross-sectional retrospective longitudinal study among Japanese people in a health program and found a novel association of higher blood UA levels with higher lung function in elderly female subjects. Moreover, genetic assaying with a UA-increasing single nucleotide polymorphism (SNP) of SLC2A9/GLUT9, or the rs11722228 T/T genotype, confirmed the impact of blood UA levels on lung function in elderly female subjects. Overall, these experimental and clinical observational analyses provide the first unique evidence that blood UA directly affects lung function in female mice under pathological conditions and in human females under aging-associated physiological conditions.In the present study, we utilized an experimental mouse model of COPD (C57/BL6J-βENaC-transgenic mice) ,27 and sWe used age-matched male and female wild-type (WT) C57/BL6J mice . All mice were maintained in the animal house at Kumamoto University in accordance with the guidelines of the animal facility center of Kumamoto University and were fed with normal chow  ad libitum. All experiments were performed according to the protocols approved by the Animal Welfare Committee of Kumamoto University No. I29-200) and all methods were performed in accordance with the relevant guidelines and regulations. Uox-KO  , origina-200 and w/v) carboxymethyl cellulose solution. Carboxymethyl cellulose sodium salt  was dissolved in distilled water for injection  and 0.5% (w/v) carboxymethyl cellulose solution as vehicle was orally administered. After 4–5 weeks of treatment, pulmonary function was measured using the flexiVent system . Mouse lungs and blood were collected 2–3 h after the last oxonate dosing. For the healthy control group, the vehicle was administered orally. To increase UA concentration in the blood, 500 mg/kg oxonic acid potassium salt in solution (oxonate)  was orally administered to 7–8-week-old wild-type (WT) and C57/BL6J-βENaC-Tg female/male mice per a day for 4 or 5 weeks. Oxonate was prepared by dissolving in 0.5%  mice were administrated intratracheally with elastase from porcine pancreas  (Sigma-Aldrich Japan) dissolved in saline . As control, saline was treated intratracheally. After 3 weeks, we measured the pulmonary function using the flexiVent system and then collected mouse lungs and blood.Measurement of pulmonary mechanics  was performed with a computer-controlled small animal ventilator flexiVent as previously described . BrieflyAfter we measured the pulmonary parameters of each mouse by flexiVent, the whole lung was filled with approximately 1 mL of 10% formalin neutral buffer solution  and then preserved in formalin for 12 h at room temperature. Samples were then washed with phosphate buffered saline (PBS) and embedded in paraffin using the histoprocessor Histos-5 . The tissue samples were separated into 3 sections, cut into sections that were 6 μm thick by a rotary microtome Leica RM2125RT , and stained with periodic acid-Schiff (PAS)  and alcian blue (pH 2.5)  to observe mucus-secreting goblet cells. After PAS and alcian blue staining, these samples were subjected to hematoxylin and eosin (H&E)  staining to visualize in light microscopy for lung morphology. The images of 10 lung sections  were captured randomly to draw 6 lines (300 μm in width) by BioRevo BZ-X710 . To determine the mean linear intercepts (MLI), the number of the intersections of 6 lines and the alveolar walls were counted and divided by the total length of the line, as previously described in detail [Blood was collected from the inferior vena cava using heparinized syringes with a 27-gauge needle. Plasma samples were obtained by centrifuging at 3000 rpm at 4 °C for 15 min and preserved at −80 °C. UA and blood urea nitrogen (BUN) in plasma were measured using DRI-CHEM . 2O2/dL). To evaluate plasma antioxidant capacity, we used the biological antioxidant potential (BAP) test (Wismer Co.), a photometric test to detect reduced iron from ferric (Fe3+) to ferrous (Fe2+). The reducing power was evaluated as the antioxidant power. The results of the BAP test were presented in μmol/L of reduced iron.To measure plasma reactive oxygen metabolites (ROMs) as an oxidative stress marker, plasma samples from mice were subjected to d-ROMs test , which measured total peroxides, including hydroperoxides (ROOH), i.e., metabolites produced when active oxygen and free radicals oxidize lipids, proteins, amino acids, and nucleic acids in the body. The unit of measurement was represented by Carratelli units “CARR U”   dye according to the manufacturer’s instructions. 16HBE14o-, A549 (1.0 × 104 cells/well), and NHBE (3200 cells/well) cells were seeded in 96-well plates and incubated until 90% confluent. NHBE was incubated with 1 nM 17β-estradiol  for 24 h before reagent treatment. After washing cells with PBS, 1.7 μM CM-H2DCFDA was added and cells were incubated for 30 min in a CO2 incubator. After 30 min, the reagent was removed and 7 or 14 mg/dL of UA  was added. NaOH at a concentration of 5 mM and 10 mM N-acetylcysteine (NAC) (Sigma-Aldrich Japan) were used as the negative and positive controls, respectively. After 30 min, cells were incubated with 100 or 400 μM H2O2 /well for 30 min and the 2′, 7′-dichloro-fluorescein (DCF) level was measured. For longer time treatment, 16HBE14o- and NHBE cells were incubated with 7 or 14 mg/dL of UA for 48 h before cells were stained with CM-H2DCFDA. The DCF level was immediately measured using a fluorescence microplate reader (excitation/emission = 485 nm/535 nm), TECAN infinite M1000 . ROS production was detected using 5(-and-6)-chloromethyl-2’,7’-dichlorodihydro-fluorescein diacetate acetyl ester . Furthermore, to investigate the association between UA and respiratory function in postmenopausal female subjects, we excluded 82 subjects under 50 years of age. Finally, the remaining 553 subjects were enrolled in this study. The study complied with the Declaration of Helsinki and was approved by the ethics committees of the Japanese Red Cross Kumamoto Health Care Center and the Faculty of Life Sciences at Kumamoto University (Genome No. 169). All subjects provided their written informed consent prior to enrollment in the study.The laboratory tests were performed using the standard methods of the Japan Society of Clinical Chemistry. The information regarding smoking habits and alcohol intake was obtained via face-to-face interviews with healthcare providers. Hypertension was defined as a systolic blood pressure (BP) of ≥140 mmHg, a diastolic BP of ≥90 mmHg, or a history of hypertension . Dyslipit-test or the Tukey–Kramer test using JMP® , as indicated in each figure legend. For correlation analysis, raw data of plasma UA and the oxidative stress marker in C57/BL6J-βENaC-Tg in untreated or oxo-treated mice were subjected to Pearson’s correlation coefficient test, also analyzed by JMP. The level of significance was set at p < 0.05. In some comparisons, effect size estimates (d) were calculated with d = 0.2 considered as a small effect, d = 0.5 as a medium effect, and d = 0.8 as a large effect.For quantitative analysis for the in vivo and in vitro studies of the mice, the data were presented as the mean ± SEM performed in indicated replicates and were analyzed by either Student’s t-test or the Mann–Whitney U test, respectively. The correlation between UA and FEV1/FVC was examined using Pearson correlation analysis. The values of UA were classified into 4 groups as follows: <5 mg/dL, 5–6 mg/dL, 6–7 mg/dL, and ≥7 mg/dL for male subjects and <4 mg/dL, 4–5 mg/dL, 5–6 mg/dL, and ≥6 mg/dL for female subjects. The associations of the classified UA levels with FEV1/FVC were examined using a multiple regression analysis with calculations of the partial regression coefficients (Bs) and standard errors (SEs). The B values were adjusted for potentially confounding factors, i.e., age and body mass index (BMI) as continuous variables and the presence of hypertension, dyslipidemia, fatty liver disease, and smoking status as nominal variables. Structural equation modeling was used to perform pathway analysis to assess the effects of UA level on respiratory function. The goodness-of-fit on the structural equation modeling was evaluated based on a goodness-of-fit index (GFI) of >0.90, an adjusted GFI (AGFI) of >0.90, and a root mean square error of approximation (RMSEA) of <0.10. A value of p < 0.05 was considered to be statistically significant. In order to examine the accuracy of the parameters of the multiple regression models and the structural equation model, bootstrap analyses were performed using 1000 replicated datasets generated by random sampling with replacement. The structural equation modeling process and the other statistical analyses were performed using the SPSS Amos software program  and the SPSS software package , respectively. For our clinical analyses, categorical variables were compared using Fisher’s exact test. Parametric or nonparametric continuous valuables were compared using Student’s Plasma UA levels in mice are notably lower than in human because mice have uricase (Uox), an enzyme that oxidizes UA to allantoin . To confp = 0.058, effect size d: 1.31; elastance, p = 0.052, effect size d: 1.35), as well as slight but not statistically significant improvement in pulmonary function parameters  compared with elastase-treated WT mice , while there was no significant difference between the oxo-treated and control groups in C57/BL6J-βENaC-Tg male mice  . Importantly, oxo treatment tended to improve FEV0.1% in female but not in male C57/BL6J-βENaC-Tg mice   and tended to increase in male mice  (r = −0.959: p < 0.001) (r = 0.746: p = 0.054)  D–E as wed: 1.02) F–H. Notad: 1.70) I. Consis< 0.001) J, but no= 0.054) K. Togeth2O2 (400 μM)-induced intracellular oxidative stress in the human bronchial epithelial cell line 16HBE14o-  in high concentrations ,11. To i6HBE14o- A, as wel6HBE14o- B. Becaus6HBE14o- , it can stradiol C.Finally, we also tested the possible detrimental effect of UA in airway epithelial cells caused by its pro-oxidative activities, as shown in other tissues ,39,40. Lp > 0.05). The SLC2A9 genotype frequencies were not significantly different between male and female subjects ) and pathophysiological conditions . A previous meta-analysis reported that hyperuricemia appeared to increase the incidence of CHD as well as the mortality of CHD patients . In addiSeveral limitations associated with the present human study should be noted. First, the human study was a retrospective design with a small number of subjects. In particular, there were few female smokers (n = 3), therefore, the association of UA with respiratory function in female smokers could not be examined. To address this limitation, we validated the findings using 1000 replicated datasets generated by random sampling of the original dataset . Since fDespite a number of reports on the detrimental effect of UA to the host due to its ability to be pro-oxidative under certain conditions, our data unveiled a direct beneficial connection between higher blood UA levels and maintenance of lung function in female humans and mice under aging-associated and/or COPD-associated physiopathological conditions. Because the idea of sex differences in disease susceptibility and drug responses is well-accepted, our finding of a novel concept whereby UA has a beneficial role in the lungs of postmenopausal elderly women and patients/mice suffering from aging-associated and/or COPD-associated pulmonary function decline contributes to the acceleration of sex-based medical study. Overall, our study demonstrated that blood UA directly affects lung function in female mice under pathological conditions and in human females under aging-associated physiological conditions. High plasma UA levels are mainly considered to be detrimental to the host due to the ability of UA to be pro-oxidative under certain conditions, which increases not only the risk of gout, but also many oxidative stress-related diseases. Therefore, our study provides the first unique evidence that blood UA levels directly affect lung function under certain conditions. Moreover, because the ideas on sex and life stage differences in disease susceptibility and drug responses are attracting great attention, our finding of a novel concept that UA has a beneficial role in the lungs of postmenopausal elderly women and patients/mice suffering from COPD contributes to the acceleration of sex-based medical study.


These receptors are thought to have mechanosensing capacity through activation of intracellular signaling via G proteins and/or the multifunctional transducer protein, β-arrestin. Importantly, ligand and mechanical stimuli can selectively activate different downstream signaling pathways to promote inotropic, cardioprotective or cardiotoxic signaling. Studies to understand how AT1R and APJ integrate ligand and mechanical stimuli to bias downstream signaling are an important and novel area for the discovery of new therapeutics for heart failure. In this review, we provide an up-to-date understanding of AT1R and APJ signaling pathways activated by ligand versus mechanical stimuli, and their effects on inotropy and adaptive/maladaptive hypertrophy. We also discuss the possibility of targeting these signaling pathways for the development of novel heart failure therapeutics.The myocardium has an intrinsic ability to sense and respond to mechanical load in order to adapt to physiological demands. Primary examples are the augmentation of myocardial contractility in response to increased ventricular filling caused by either increased venous return (Frank–Starling law) or aortic resistance to ejection (the Anrep effect). Sustained mechanical overload, however, can induce pathological hypertrophy and dysfunction, resulting in heart failure and arrhythmias. It has been proposed that angiotensin II type 1 receptor (AT Indeed, Rockman’s group recently revealed that gene deletion or selective inhibition of AT1R in mouse hearts abrogates the Frank–Starling relationship  production/release, which in turn activates endothelin type A receptor (ETAR) to induce transactivation of epidermal growth factor receptor (EGFR) through ROS-induced ROS release . Cingolani’s group has described a similar complex signaling pathway upon stretch-induced Ang II production, involving autocrine/paracrine activation of AT1R and ETAR  signaling pathway   6 channel is an additional important upstream determinant of SFR. Our study demonstrated that gene deletion and selective blockade of TRPC6 channel abrogates SFR and the Anrep effect in physiologically loaded myocytes, muscle strips, and intact hearts . More re pathway . Indeed,hibition . Ca2+ ins ERK1/2  upstream via NCX   . Despites (ARBs) , mechaniN-terminal kinase  signaling cascade , c-Jun Nl kinase , Akt–maml kinase  and calcl kinase . In partl kinase . Once actor NFAT . It has C3 and 6 . Importamulation . In addimulation . Althoug1R activation by either Ang II or mechanical stress not only induces pathological signaling but also promotes physiological hypertrophy and prosurvival signaling. In embryonic, neonatal, and adult cardiomyocytes, Ang II–AT1R activation promotes transactivation of EGFR, which in turn activates MAPK and Akt–mTOR pathways  that is required for the recruitment of β-arrestin2 and activation of downstream signaling pathways, such as EGFR transactivation and ERK phosphorylation (αi proteins primarily inhibit the cAMP-dependent pathway by inhibiting adenylyl cyclase (AC) activity. Although previous studies have shown that Ang II may promote AT1R–Gαi coupling to inhibit AC and to regulate Ca2+ channels in certain tissues or cell types , TRV120023, TRV120027, and TRV120067 have been shown to selectively activate β-arrestin-mediated pathways and therefore termed as β-arrestin-biased agonists  and TRPC3/6, could be another therapeutic strategy. Our study previously revealed that adverse myocardial responses induced by mechanical stimulation to these channels are suppressed by post-transcriptional modification of TRPC6 channel by activation of the cGMP–PKG pathway  . Increas stretch . Our stutivation  , as controlled by PKC-directed phosphorylation , which may contribute to the pro-hypertrophic program  and molecular modeling approach, 1R induces a dislocation and a counterclockwise rotation of transmembrane domain 7 toward the ligand-binding pocket, a conformation that is distinct from the ligand-activated receptor conformation. These fascinating data suggest that conformational change is responsible for biased signaling in AT1R. Such studies of APJ and examination of the differential effect of novel AT1R ligands on its conformation could eventually lead to structure-based drug design focused on selective inhibition of adverse remodeling via stretch-dependent pathways.G protein-coupled receptors adopt distinct conformations to selectively activate different arrays of downstream signaling . Detaileruitment . More reruitment . Another1R and APJ systems has been well-studied. Infusion of Ang II in rats causes an acute reduction of apelin and APJ levels in the heart, which is abolished by treatment with an ARB (1R gene deletion (1R and APJ systems through neurohormonal interactions, direct physical interaction between AT1R and APJ has also been reported. Co-immunoprecipitation and FRET analysis revealed heterodimerization of AT1R and APJ, in which the interaction appears to induce the inhibition of the AT1R signaling pathway (1R–APJ heterodimerization is induced by apelin, but is not affected by Ang II (1R–APJ have not been examined. It is conceivable that AT1R–APJ heterodimers activate distinct signaling pathways compared to monomeric receptors, or that the multiple reported downstream pathways of AT1R and APJ may be partially dependent on the activation of the heterodimeric receptor. Understanding of this may lead to the identification of novel ligands with optimal effects on heart failure pathobiology.Neurohormonal interaction between ATh an ARB . The lefdeletion . Furtherdeletion . While a pathway . More rey Ang II . To date1R and APJ signaling to identify novel therapeutic strategies. Selective activation or inhibition of mechanically stimulated signaling components by biased agonists may yield more precise molecular enhancers of desired inotropic or cardioprotective effects while avoiding detrimental signaling. In addition, by gaining an improved understanding of the signaling mechanisms of these receptors in the heart, it is likely that complementary downstream targets will be identified to modulate cardiac function and ameliorate disease.Angiotensin II type 1 receptor and APJ are mechanosensitive GPCRs in the heart, playing vital roles in cardiac physiological adaptation to changes in mechanical load. However, when a mechanical load is excessive and sustained, it can induce maladaptive hypertrophic signaling. Enormous effort has been invested in understanding the physiological and pathophysiological roles of ATKS took the lead in writing the manuscript with input from VP and EA.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To prepare a porcine model of obstructive sleep apnea-hypopnea syndrome (OSAHS) and observe the pathological and hemodynamic changes in the common carotid artery. Twelve male miniature pigs were randomly divided into the model and control group (n = 6). Pigs in the model group were kept in an air-flow negative pressure chamber at 0.96 ± 0.01 kPa, and the air oxygen content, temperature, and humidity were kept at normal culture conditions in both groups. After pigs in the model group presented symptoms of OSAHS, changes in the hemodynamics and morphology of the carotid artery were analyzed using color Doppler, and light and electron microscopy. An animal model of OSAHS was successfully created. The internal diameter of the carotid artery of pigs in the model group was decreased, while the intima thickness, peak-systolic mean velocity, and resistance index were increased when compared to the control group (P < 0.05). The results of the light and electron microscopy revealed an incomplete elastic plate, increased media thickness, irregular morphology of the smooth muscle cells, increased collagen fiber bundles, partially disordered elastic fibers, and smooth muscle layers. The quantitative analysis showed significantly increased elastic fibers in the media of the carotid artery in the model group (P < 0.01). Pathological changes in the tissue structure and hemodynamics in the negative pressure-induced pig OSAHS model were observed. We suggest that alterations in the upper airway pressure during OSAHS may lead to cardiovascular conditions through its pathological effects on the carotid artery. Obstructive sleep apnea-hypopnea syndrome (OSAHS) is characterized by repetitive episodes of airway reduction (hypopnea) or cessation (apnea) due to pharyngeal narrowing, leading to fragmented sleep cycles [u293a]. It was found that those who were male, of advanced age, and had higher body mass index had a higher prevalence of OSAHS [2]. Studies have looked at the aerodynamic properties of the pharynx in OSAHS, and a greater negative pressure in the pharyngeal cavity was shown to contribute to increased collapsibility of the pharynx [3]. Additionally, the pharynx has smaller average area and more compliance in OSAHS, leading to a narrower and more collapsible upper airway in OSAHS patients [4]. It has also been found that an increase of 1 unit in sleep apnea-hyponea index increased the risk of stroke by 6% in men [5]. Independent studies have shown that patients with OSAHS are at increased risk of ischemic heart disease, atherosclerotic coronary disease, diabetes, hypertension, congestive heart failure, acute coronary syndrome, stroke, and cardiovascular mortality [u293d]. Moreover, OSAHS patients have increased frequency of stroke and arterial fibrillation [10]. The application of continuous positive airway pressure to increase air pressure in the throat and prevent its collapse could reverse or attenuate OSA and its associated cardiovascular effects [u293e]. However, the mechanism of how OSAHS affects the cardiovascular system is still unclear.Previous studies used rat models to investigate the effect of OSAHS on the cerebral cortex, skeletal muscle, and visceral fat tissues [u293f]. Studies have also employed a porcine model system to study the effect of OSAHS on the progression of chronic kidney disease [u2940]. As the size and cardiac physiology of porcines are similar to those in humans, this model system confers a huge advantage over other systems [u2941]. In this study, we attempted to probe the link between cardiovascular system and OSAHS by measuring changes in the structure and hemodynamic properties of the carotid artery during OSAHS in a porcine model. We measured several parameters, including the thickness of the intima, inner diameter of the carotid artery, peak-systolic mean velocity (S), and resistance index (RI) [15]. We investigated if the differences in the structure and hemodynamic properties of the carotid artery in OSAHS and normal cases could contribute to the development of cardiovascular conditions.A total of 12 male miniature pigs, aged 6 months and weighing 17 ± 1.5 kg, from Guizhou Province, Southwest China, were used. After habituation, the 12 pigs were randomly and equally divided into the model and normal control groups. All of the procedures and animal experiments were approved by the Animal Care and Use Committee of Jiangsu University.A negative pressure chamber , measuring 1.5 × 2 × 3 m, was made of metal sandwich foam material, and the atmospheric pressure was kept at 0.96 ± 0.01 kPa. The air oxygen content was maintained at 22.5 ± 0.3%, and the temperature and humidity were maintained at 22–24 °C and 40%–50%.After 3 months of adaptive feeding, pigs in the model group were placed and fed in the negative pressure chamber for 6 months  to observe the activities, sleep, and snore decibel of the animals. The respiratory rate, respiratory waveform, and respiratory pressure were detected using the mechanical characteristics of the airway to assess if the pigs in the model group had symptoms similar to those of an OSAHS patient. The normal control group was fed in a similar chamber with the same conditions, except for a low negative pressure.Pigs in the model and control groups were anesthetized by an intramuscular injection of ketamine . The porcine carotid artery diameter, intima-media thickness, S, diastolic end blood velocity (D), RI, carotid artery peak systolic velocity, and diastolic end blood flow velocity ratio (S/D) were measured using a United States Acuson-XP/10 EEG Doppler diagnostic instrument . The measurement site was located at the bifurcation of the common carotid artery.The miniature pigs were euthanized via carotid artery bleeding. The left carotid artery of each experimental pig was taken, 10 g of the fresh sample was fixed with 10% neutral formaldehyde solution , and the paraffin sections were prepared. The left carotid artery structure was observed using a PUS-BX51 OLYM optical microscope . Using an HPIAS-1000 high definition color pathology image analysis system, the film thickness of the common carotid artery and the elastic fibers were quantitatively analyzed.Another fresh sample of 1 cm3 was fixed with 2.5% cold glutaraldehyde (Sinopharm) and then 1% osmium tetroxide (Sinopharm) for 2 and 1 h, respectively. The specimens were dehydrated with graded acetone , embedded with resin Epon-812 , and located by semithin sectioning for 70-nm ultrathin sections. The sections were observed under a JEM-1200 transmission electron microscope after uranyl acetate (Sinopharm) and lead citrate double electron staining.Data were processed and analyzed using SPSS 10  statistical software. The data were represented as the mean ± standard deviation. T test was used to compare the model and control groups. P < 0.05 indicated statistical significance.After eating and staying in the hypobaric chamber for 6 months, there was no significant difference in body weight with the same amount of diet between the control and the model groups . However, the miniature pigs in the model group showed reduced activity, increased sleeping time, snoring (43–51 dB), accelerated respiratory rate, negative respiratory pressure value (3.5 to 4 kPa), and an irregular pattern of respiratory pressure waveform, similar to OSAHS symptoms in humans . The respiratory pressure waveform in the control group was normal , indicating that the experimental group had OSAHS and could be used as a model system to investigate it.The intima of the common carotid artery visualized using 2-dimensional ultrasound was smooth in the OSAHS pigs. The medial layer had low echo and reflectivity. The internal diameter of the common carotid artery was smaller, and its intimal thickening was significantly higher than in the control group (P < 0.05). The hemodynamic parameters of the carotid artery in the model group were measured by Doppler imaging. All 3 parameters  were increased in the pigs with OSAHS, indicating increased vascular resistance when compared to the control group (P < 0.05) (Table 1)In the model group, the common carotid artery wall was thicker, had poorer elasticity, greater toughness, and greater puncture resistance. In the control group, the artery tube wall was uniform, had good elasticity, punctured smoothly without resistance, and had no obvious lesions. Pigs in the model group had an artery tube wall thickness of 655.16 ± 35.59 µm. The inner layer of the carotid artery had irregular and sparse elastic fibers, and an incomplete internal elastic lamina . The film thickness was 525.34 ± 49.82 µm, layered yet clear, had smooth muscle cells, and the nuclear morphology was irregular. The hematoxylin and eosin (H&E) staining showed the proliferation and disordered arrangement of the elastic fibers . In the control group, the arterial wall thickness was 561.53 ± 44.98 µm and the carotid artery wall had a 3-layer structure, with the inner layer consisting of monolayer of endothelial cells, connective tissue, and internal elastic lamina. The endothelial cell morphology was normal and the elastic plate had a continuous wave shape . The film thickness was 410.16 ± 23.76 µm, medial smooth muscle, and the elastic fibers had an ordered arrangement ; the external elastic plate was not obvious. The quantitative analysis of the elastic fibers (Table 2) showed that the total number of the elastic fibers, their surface number density, total area, and area percentage in the left carotid artery in the model group were significantly higher than those in the control group (P < 0.01).Electron microscopy showed that the carotid endarterium had no obvious change, the collagen fiber bundles were increased, some elastic fibers and smooth muscles of the layered structure were disordered, the fibrinoid materials were focally distributed, and the smooth muscle cells had irregular morphology . Moreover, the cell filaments were reduced, the nucleus was irregular, and red blood cell extravasation was visible near the endometrium. Electron microscopy of the control group showed that the morphology of the smooth muscle cell surface was rich with irregular cellular protrusions. There was abundant cytoplasm, a high content of myofilaments, clear macula densa, increased interstitial collagen fibers, and no observed inflammatory cell infiltration . In the model group, hyperplasia, degeneration, and focal fibrinoid necrosis were found in the medial elastic fiber of the left carotid artery . Degeneration and disordered arrangement was found in the medial smooth muscle of the left carotid artery . For a comparison of the general morphology, the light and electron microscope observations of the left carotid artery wall in the control and model groups are shown in Table S1.In this study, we investigated the link between OSAHS and cardiovascular conditions associated with it. To that end, we assessed the differences in the structure and hemodynamic properties of the carotid artery in miniature pigs with OSAHS and control pigs. It was found that the internal diameter of the common carotid artery in the upper airway of the mini pigs with OSAHS was smaller, its elasticity was reduced, the intimal thickening was increased, and the elastic fibers were disordered. It was shown that the morphology of the smooth muscle cells in the left carotid artery was irregular with a focal deposition of the fibrinoid material in the model group. It was also shown that the hemodynamic parameters, including the D, RI, and ratio of systolic and diastolic pressure were increased in the model group. Thus, OSA and hypopnea caused by the upper airway obstruction lead to changes in the structure and hemodynamic behavior of the carotid artery during OSAHS. Previous studies have shown that assessment of the carotid artery is an important index for estimating the risk associated with coronary artery diseases [u2942]. Thus, we suggest that these changes in the carotid artery could explain the emergence of cardiovascular diseases.Clinical studies have found endothelial dysfunction and arterial wall hardening in OSAHS [u2943], which are different from the physiological degeneration or other conditions caused by arterial diseases [u2944]. However, there is still a lack of relevant research regarding the effects of OSAHS on the cardiovascular system of patients. In this study, we successfully prepared an OSAHS mini pig model with the application of a negative pressure chamber, which has been previously shown to cause OSAHS [u2945]. We used the miniature pig model system because the coronary circulation and arterial anatomy of pigs and humans are very similar. Moreover, pigs offer the best model to study plaque instability, as they have a similar lipoprotein profile to that of humans [u29460]. Abnormal pharyngeal airway pressure is one of the critical factors in the pathogenesis of OSAHS. When the upper airway is in a negative pressure state, the soft tissue of the pharyngeal cavity gradually undergoes hyperplasia, hypertrophy, and reconstruction, causing the pharyngeal cavity to narrow and reduce the effective respiratory cross section area. This causes a further lack of oxygen, promoting OSAHS formation and development [7]. Studies have also enabled a reversal of the symptoms of OSAHS by either changing the sleeping position or by the application of a continuous positive airway treatment [u29471]. Previous studies housed SD rats in sealed cabins in a hypoxic environment, leading to reconstruction of the soft palate and snoring . It was found that the structure and function of the pharynx in rats was similar to the pathological changes of OSAHS after the induction of OSAHS by low pressure hypoxia [u294a]. In this study, we constructed an OSAHS model of Guizhou miniature pigs using a controlled low pressure module and observed the hemodynamic and histopathological characteristics of the common carotid artery. We also showed that the symptoms of the pig model in the low pressure chamber environment were similar to those of OSAHS; thus, miniature pigs can be employed as an effective OSAHS disease model.However, we only observed the hemodynamics and pathological changes of the common carotid artery, and OSAHS is a systemic disease, where long-term hypoxia only causes damage to the cardiovascular system, triggering atherosclerosis, sudden death, cardiovascular and cerebrovascular accidents, hypertension, and other diseases, but it can also lead to neurological damage, such as nerve cell stress response, apoptosis, and degeneration. Furthermore, our observations were limited to changes in the vascular morphology, and the potential molecular signaling pathways were not further discussed. Further studies are required to separate the vascular endothelial cells and smooth muscle cells in the experimental and control group to detect the differential expression of mRNA, miRNA, lncRNA, or protein, and verify the downstream molecular targets. Meanwhile, the evidence also showed that gut microbiota contributed to the hypertension observed in OSA [u294b], this indicated that gut microbiota may have been an influential factor in inducing OSAHS diseases. However, the study also showed that OSAHS could alter gut microbiota [u294c]; hence, future experiments could be designed to verify the direct causal relationship between gut microbiota with OSAHS.In this study, the structure and hemodynamic characteristics of the carotid artery in mini pigs with OSAHS and a control were compared to investigate the link between cardiovascular diseases and OSAHS. It was shown that miniature pigs can be employed as a model system for OSAHS by keeping them in low pressure chamber, which lead to the reconstruction of the upper airway and thus, OSA and hypopnea. It was also observed that the common carotid artery in the upper airway of mini pigs with OSAHS had a thinner internal diameter, reduced elasticity, disordered elastic fibers, and increased intimal thickening. The hemodynamic parameters of the carotid artery  were also increased. We speculated that these changes in the carotid artery properties could lead to increased cardiovascular conditions. Thus, this study provides a link between cardiovascular diseases and OSAHS; however, further studies are required to identify the downstream molecular targets of inducing negative pressure in the upper airway.This study was supported by the National Natural Science Foundation of China (11472118), the Maternal and Child Health Research Project of Jiangsu Province (F201604), and the Science and Technology Development Fund of Wuxi City (WX18IIAN044).


Drosophila genome for modifiers specific to either weak or strong Ras-driven eye phenotypes. Our screen enriched for regions of the genome not previously connected with Ras phenotypic modification. We mapped the underlying gene from one modifier to the ribosomal gene RpS21. In multiple contexts, we show that RpS21 preferentially influences weak Ras/MAPK signaling outputs. These data show that codon usage manipulation can identify new, output-specific signaling regulators, and identify RpS21 as an in vivo Ras/MAPK phenotypic regulator.Signal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. Here, by altering codon usage as a novel platform to control signaling output, we screened the  Cellular communication is critical in controlling the growth of organs and must be carefully regulated to prevent disease. The Ras signaling pathway is frequently used for cellular communication of tissue growth regulation but can operate at different signaling strengths. Here, we used a novel strategy to identify genes that specifically tune weak or strong Ras signaling states. We find that the gene RpS21 preferentially tunes weak Ras signaling states. These same pathways can be aberrantly regulated in disease. Large numbers of molecular regulators of these pathways have been identified using high-throughput genetic screening. Additionally, quantitative imaging approaches have revealed intricate signaling regulation. This regulation includes feedback control of the duration or strength of a downstream biochemical signaling output . A current challenge is to place the numerous identified signaling pathway regulators in the context of complex signaling dynamics, and to relate such regulation to Drosophila eye [C. elegans vulva [Drosophila cell-based screens using a biochemical MAPK output (Erk phosphorylation) have identified many other Ras/MAPK regulators [An example of the complexity of signaling regulation is the evolutionarily conserved Ras/mitogen activated protein kinase (MAPK) pathway . In canohila eye –9 and C.ns vulva –14, regugulators –17.in vivo context plays a role in whether a given strength of signaling output leads to a phenotypic output. Specifically, taking a similar optogenetic approach in the early developing fly embryo, it was shown that manipulating Erk activation strength has minimal effects on cells at the poles of the embryo, but has a profound impact on development of cells in the middle of the embryo [Drosophila or zebrafish was recently shown to either activate or repress Erk phosphorylation depending on the cell type and gene expression environment [in vivo phenotype.These numerous molecular regulators contribute to a diversity in Ras/MAPK signaling dynamics. Using an optogenetics-driven MAPK activation approach in cultured mammalian cells, it was revealed that distinct Ras/MAPK regulation (such as a paracrine STAT3 circuit) can distinguish between biochemical signaling outputs, namely sustained (strong) or transient (weak) Erk activation by Ras . These be embryo . Furtherironment . The degironment ,23. ThesDrosophila Ras gene [Ras85D, hereafter Ras). Rare codons are well-associated with poor mRNA translation [KRAS to their common counterparts leads to elevated translation, protein, signaling, and transformation [Ras transgene. We then report the use of such transgenic flies to screen a whole genome deficiency (termed Df for convenience) kit for genetic modifiers of eye phenotypes that are specific to only strong or only weak Ras/MAPK signaling. Our screen specifically looked for modifiers unique to specific Ras signaling states, by leveraging the differential signaling phenotypic output driven by rare versus common codons in the Ras gene. Importantly, the Ras gene enriched in rare codons used in our screen models more closely the rare codon-enriched sequence of human KRAS [Here, we introduce a novel approach to genetically screen for signal output-specific regulators of Ras/MAPK signaling. This approach, which should be applicable to any signaling pathway, involves controlling the amount of active Ras protein produced by changing codon usage in the single Ras gene  BSC692, an enhancer of the rough-eye phenotype driven only by weak Ras/MAPK signaling, to the ribosomal protein S21 gene (RpS21). We show that RpS21 negatively regulates Ras protein levels in several contexts, the effect of which is preferentially manifested at low levels of MAPK signaling. This approach highlights the usefulness of codon manipulation as a viable approach to identify signal output-specific signaling regulation and introduces new genetic reagents to explore weak Ras signaling regulation in Drosophila. Our uniquely identified modifiers include those specific to Ras with rare codons, like that of human KRAS.Our screen enriched for genomic regions not previously ascribed to Ras phenotypic modification. Of the 15 Drosophila Ras transgene (termed RasV12 here for convenience). To control MAPK signaling strength during fly development, we opted for the new approach of simply changing the codon usage of a RasV12 transgene. Codons that occur infrequently in a given genome (rare codons) are known to impede translation, including in Drosophila [Drosophila codon usage (see Drosophila Ras transgenes: 1) we altered none of the codons (RasV12Native), 2) we made all codons the most commonly occurring in the genome (RasV12Common), 3) we made all codons the most rare in the genome (RasV12Rare), and 4) we created a control wild-type version lacking the V12 mutation and also lacking codon alteration (RasWTNative). To monitor expression, all four transgenes were epitope-tagged at the N-terminus with a 3XFLAG-tag sequence and expressed under the control of a Gal4-inducible UAS promoter . To control for position effects, all transgenes were integrated at the same site in the genome .To identify Ras/MAPK molecular regulators that differentially impact strong or weak signaling outputs, we required a platform to tightly control the strength of MAPK signaling. To activate the pathway, we expressed a highly conserved, mutant active (G12V) osophila –35. By eosophila ,37. Thissage see  and creadex (CAI ) to RasV12Common , while tnome see . Our altin vivo phenotypic readout rather than a biochemical readout, an approach validated by quantitative studies of MAPK activation in Drosophila embryos [Drosophila eye is a highly accessible model. Driving expression of RasV12 in the developing eye with an eye-specific promoter such as sevenless (sev) dysregulates the proper differentiation of the R7 photoreceptor cell, leading to an easily scored ‘rough-eye’ phenotype [To measure signaling output strength of our transgenes, we first chose to use an  embryos ,21. For henotype ,40. Thishenotype .Ras transgene in vivo by driving their expression in the developing fly eye using sevenless (sev)-Gal4. As expected [RasWTNative in this manner does not result in a rough-eye phenotype . However, when we expressed the constitutive-active versions of Ras (RasV12), we found a range of rough-eye phenotypes . We binned these phenotypes into one of three classes: severe, moderate, or mild. Each class was assigned an increasing numeric score, based on the incidence and severity of eye phenotypes such as necrotic spots and discoloration . To determine whether Ras protein levels track with the difference in Ras-driven rough-eye phenotype, we isolated heads from flies encoding common and rare RasV12 transgenes and performed serial dilution immunoblotting with an anti-FLAG antibody . Separately, flies expressing all three active Ras transgenes were again immunoblotted with an anti-FLAG antibody, and protein levels were normalized to a loading control . In both experiments, we found RasV12Common flies express roughly 1.5 to 1.9-fold more Ras protein than flies expressing RasV12Rare . Additionally, RasV12 protein levels are similar between RasV12Native and RasV12Common flies , which is consistent with the similar codon content between these transgenes . These experiments established that codon usage can be manipulated to examine an in vivo, Ras signal-driven output (eye phenotype), and identified both weak (RasV12Rare) and strong (RasV12Common) versions of this output. Further, these results are consistent with RasV12Rare serving as a model of the rare codon bias of human KRAS, the most commonly mutated RAS family member in human cancers.We assayed the phenotypic output of each expected , expression  see . We thenRas gene on Ras signaling and Ras GTPase activity. To examine the effect of expression of RasV12Rare versus RasV12Common transgenes on MAPK signaling, we measured the level of phosphorylated Mek  and Erk  compared to the total level of these proteins by immunoblot analysis. RasV12Common animals exhibit elevated levels of p-Erk and p-MEK compared to RasV12Rare fly heads . We independently verified this difference in cultured S2 and KC insect cells . Further, using a Ras binding domain (RDB) pull-down assay [RasV12Common contain a higher total level of active Ras than cells expressing RasV12Rare . In sum, our findings establish RasV12Rare and RasV12Common as two distinct transgenes that either weakly or strongly activate Ras/MAPK signaling output , and that transgene-driven signal strength tracks with an observable difference in phenotypic output.We next assessed the impact of codon content in the ells see , again fwn assay , we founRasV12Common mRNA is significantly higher than RasV12Rare in adult heads . These findings are consistent with the model that altering codon usage of Drosophila Ras impacts Ras RNA, Ras protein, and Erk signaling.Codon bias has been shown to impact not only translation fidelity and efficiency –47 but aRasV12Rare and RasV12Common at single cell resolution. Using the FLP-out system [Tubulin-Gal4 driver. Clones generated in this system are marked with GFP. We used heat shock to control the frequency of FLP-out events, and used a level of heat shock that resulted in 1–2 discrete clones in leg imaginal discs . We note that RasV12 expression is connected to animal lethality in other contexts, including when induced transiently or in somatic clones [RasV12Rare significantly increases clone size relative to controls , which is consistent with the well-known role of Ras/Erk signaling in promoting cell proliferation. Interestingly, in surviving RasV12Common animals, clones are no bigger than in RasNative controls . Taken together with the organismal death and frequent necrotic spots seen in the eyes of sev-Gal4, UAS-RasV12Common animals, we interpret this result to likely reflect the increased apoptosis or cellular senescence that can result from increased Ras expression [RasV12Rare and RasV12Common have such differing effects on cell proliferation in leg discs, our results underscore the critical importance of signal output levels on Ras-driven phenotypes and highlight that lower Ras levels can actually drive more cell proliferation in specific contexts.We next examined the impact of t system , we geneiscs see . In thesc clones –57. In spression ,59. GiveRasV12Common and RasV12Rare rough-eye phenotypes were both in the range that can be modified. Specifically, two different heterozygous loss-of-function mutations known to suppress active Ras phenotypes, namely the S-627 allele of kinasesuppressor ofras, (FlyBase: ksr) [S-2554 allele of beta subunit of type Igeranylgeranyltransferase, (FlyBase: betaggt-I) [RasV12Common and RasV12Rare . Next, we examined heterozygous mutants of the yan-XE18 allele of anterioropen, or aop, which is known to enhance the active Ras phenotype [aopyan-XE18/+, we did observe a marked decrease in another phenotypic readout- animal survival. As for our FLP-out experiments with Tubulin-Gal4, sev-Gal4 expression of RasV12Common leads to considerably more organismal death than with RasV12Rare . This sev-Gal4-driven lethality likely reflects the expression of sevenless-Gal4 in other tissues [aopyan-XE18/+ animals expressing both RasV12Common and RasV12Rare transgenes . These results establish that codon-altered RasV12 transgenes are subject to phenotypic modification, including by dose-sensitive heterozygous mutations.We next sought to use our codon alteration system to gain insight into how the Ras/MAPK pathway can be differentially regulated in different signal-strength states. To do so, we screened for molecular regulators that modify Ras/MAPK phenotypes driven only by strong or only by weak signaling states. We first confirmed that se: ksr) , and thetaggt-I) . As withhenotype ,61 AlthoRas cDNA to express activated Ras [S1B Fig) and is similar to RasV12Common in terms of MAPK biochemical and phenotypic outputs . To find unidentified modifiers that may be specific to weaker (or stronger) Ras/MAPK-driven phenotypes, we conducted a genome-wide unbiased heterozygous mutant screen to specifically identify modifiers of the rough-eye phenotype driven by only RasV12Rare, (or only RasV12Common), . We used the Bloomington Deficiency (Df) Kit, which covers 98.3% of the euchromatic genome. In a primary screen (S1 Table), we crossed 470 Dfs representing 99.1% of the Df collection to animals with RasV12Rare or RasV12Common expressed in the eye by sev-Gal4, and scored the resulting eye severity in an average of 30 (RasV12Common) or 60 (RasV12Rare) progeny animals per cross. We also factored animal lethality into our scoring . Interestingly, we identified more enhancers than suppressors . The reason for this remains to be determined, but we note that our calculation of phenotypic modification , meaning they scored as only modifying the eye phenotype driven by a single signaling state . Using a low-stringency cutoff score (see Dfs in our primary screen (S1 Table). To filter our hits to those that were the most robust, these Dfs were then re-tested in a secondary screen  by crossing them a second time to sev-RasV12Common and sev-RasV12Rare. In this screen, we used a more stringent cutoff score to ensure repeatability to define a robust differential modifier , that reproducibly differentially modify either only RasV12Common or only RasV12Rare . Among these differential modifiers, we again recovered more enhancers than suppressors, although importantly we recovered both enhancers of RasV12Common and suppressors of RasV12Rare, arguing that our screen had the dynamic range to modify both strong (RasV12Common) and weak (RasV12Rare) Ras/MAPK signaling outputs .As expected, we found general tion see  includedcore see , we idenfier see . This scRas genetic enhancers and suppressors  do not encompass known Ras modifiers, supporting the idea that our signal strength-specific modifier hits are enriched in new Ras enhancers and suppressors . To explore possible relationships amongst these 15 differential modifier Dfs, we queried the genes within differential versus enhancer and suppressor Dfs against the established list of FlyBase Gene Groups (FBGG). Interestingly, the gene groups enriched in the differential Dfs do not overlap with those in the general enhancer/suppressor Dfs , suggesting that the differential modifiers may represent a distinct class of Ras modifiers. Unlike the general modifier Dfs, differential modifier regions are enriched for basic Helix Loop Helix (bHLH) transcription factors, potentially reinforcing their distinct regulation of Ras/MAPK signaling. In summary, by controlling Ras/MAPK signal output strength through codon usage and using a phenotypic output screen, we successfully identified Dfs that alter a Ras/MAPK phenotype in a signaling output-specific fashion.We next queried both the general  and differential  modifiers against a FlyBase database of all reported sors see . 56% of Df(2L)BSC692 as it was one of the smallest deficiencies, encompassing only 12 genes, that specifically enhanced RasV12Rare . Of these 12 genes, RibosomalproteinS21, or RpS21 , represented a plausible candidate modifier. RpS21 stands out among small ribosomal subunits for its reported negative regulation of hematopoietic and imaginal disc hyperplasia [RpS21 is a responsible gene in Df(2L)BSC692 for specifically enhancing RasV12Rare, we assessed the rough-eye phenotype of RasV12Common and RasV12Rare in the background of the mutant RpS2103575. Indeed, only the sev-RasV12Rare rough-eye phenotype is enhanced in the RpS2103575/+ background . RpS2103575/+ did not score as a hit by our animal lethality criteria . However, cn mutations were also present in 4 other Dfs in our screen, only one of which was a hit. Therefore, RpS21 and not cn is the likely modifier on the RpS2103575 mutant chromosome. Similar to our findings in the eye, RpS2103575/+ preferentially impacts the phenotype of RasV12rare leg imaginal disc clones. We observe smaller average clone sizes in RpS2103575/+, RasV12Rare animals relative to RasV12Rare alone, whereas RpS2103575/+ does not impact clone size in RasV12Common animals . Together, these findings identify RpS21 as a responsible modifier of RasV12Rare in one Df from our Ras/MAPK signal strength-specific screen.To identify a differential modifier from our screen at the single gene level, we focused on erplasia . To deteeria see , suggestRpS21 mutant allele and a small deficiency encompassing this gene (Df(2L)BSC692) as differential RasV12 eye phenotype modifiers. We next examined the molecular alterations of Ras signaling that underlie this signal intensity-specific modification. To this end, we assessed RasV12 levels and/or MAPK pathway activation by immunoblot analysis in three distinct cellular and signal output settings: ectopic Ras activation in adult fly heads, ectopic Ras activation in cultured S2 cells, and endogenous MAPK signaling in ovaries. Our results overall show that while RpS21 reduction impacts Ras/MAPK in numerous settings, there is a more pronounced effect in cases where signaling output is weaker.From our genome-wide screen and follow-up mapping efforts, we were able to identify both an RasV12Rare flies, transgenic Ras protein levels increase in RpS2103575/+ animals relative to wild type. This result is consistent with the enhanced RasV12Rare eye phenotype in RpS2103575/+ animals. However, unlike our lack of an observable phenotypic enhancement of RasV12Common in the eye, at the biochemical output level we also observe an increase in the level of RasV12Common in the RpS2103575/+ background . This result shows that RpS2103575 modifies both sevenless-driven RasV12Rare and RasV12Common protein levels in the adult fly head, but only RasV12Rare modification leads to an observable phenotypic output in this setting. This difference between eye phenotype and protein level effects could suggest that a large difference in Ras protein change is needed to cause a detectable change at the eye phenotype level. Alternatively, our adult head assay focuses on Ras levels in the adult animal, whereas our eye assay focuses on the effect of RpS21 reduction during eye development. RpS2103575/+ does not impact RasV12Rare or RasV12Common RNA levels in adult heads, suggesting RpS21 acts at the translational level to impact Ras signaling .In the heads of RasV12Common or RasV12Rare, and then used RNAi to reduce RpS21 levels. As in the fly head, RpS21RNAi elevates RasV12Rare protein levels. However, unlike in the head, RasV12Common protein levels in S2 cells are unaffected by RpS21RNAi . We note that, in these cells, our expression system led to particularly robust expression of the RasV12Common protein . We also examined MAPK activation in S2 cells. Whereas p-Mek and p-Erk are noticeably increased in RpS21RNAi S2 cells expressing RasV12Rare, we see no overt increase in these MAPK activation readouts upon RpS21RNAi in S2 cells expressing RasV12Common . Taken together, our results in the head and in S2 cells suggest that when Ras signaling is above a particular threshold , RpS21 reduction does not impact pathway output.Next, we examined the impact of RpS21 on Ras signaling in additional cellular contexts. We first transduced S2 cells with an expression vector encoding either in vivo. To do so, we examined the effect of disrupting one allele of the RpS21 gene on endogenous MAPK signaling in the ovaries of flies, a tissue where EGFR/Erk signaling has a well-defined role [Fig 4G). Of note, RpS2103575/+ animals have no obvious female fertility defects. In this tissue, endogenous p-Mek and p-Erk levels increase in both Df(2L)BSC692/+ and RpS2103575/+ animals relative to control w1118 animals . Although we were not able to successfully determine endogenous Ras levels in the ovary with existing reagents (not shown), our overall findings are consistent with RpS21 negatively regulating endogenous Ras/MAPK signaling in this tissue. Collectively, we find that loss of RpS21 elevates Ras/MAPK signaling in multiple contexts.We also assessed whether endogenous MAPK signaling can be regulated by RpS21 ned role ,67 and wRasV12Common expression should render fly eye development sensitive to the RpS2103575/+ mutant background. To experimentally test this threshold model, we took advantage of the well-known fact that expression of transgenes using the Gal4-UAS system is responsive to temperature, with higher temperature resulting in higher expression over the physiological range of 18°C-29°C. We thus evaluated the rough-eye phenotype of sev-RasV12Common versus sev-RasV12Rare flies in a wild-type versus RpS2103575/+ mutant background, only this time at 18°C. At this lower temperature, RpS2103575/+ now acts as an enhancer of RasV12Common . Interestingly, RpS2103575/+ no longer enhances RasV12Rare, underscoring the sensitivity of RpS21/+ to Ras/MAPK signaling strength. Therefore, RpS21 regulation of the Ras pathway appears to be signal-strength dependent, rather than codon-dependent. Collectively, these results demonstrate that while RpS21 negatively regulates Ras-MAPK signaling in diverse contexts, at the phenotypic level this regulation preferentially impacts weak Ras/MAPK signaling. These findings are consistent with a model whereby above a certain signaling intensity threshold, regulators that impact Ras signaling at weaker intensity levels are no longer effective .Our immunoblot analysis validates our genetic screen finding that RpS21 can negatively regulate Ras and/or MAPK signaling, in a manner that potentially depends on the strength of Ras/MAPK signaling. One interpretation of these data is that RpS21 has a minimal effect on MAPK signaling output above a certain threshold of MAPK signaling. Such a model would predict that experimentally reducing the amount of ubiquitin promoter and were integrated into the same site in the genome . Given this, we next tested whether RpS21 downregulation alters GFP protein expression in a codon-dependent manner. RpS2103575/+ animals exhibit similar GFPRare protein expression as wild type animals . Additionally, RpS2103575/+ animals exhibit similar GFPCommon protein expression as wild type animals . These results indicate that RpS21 downregulation does not impact translation of at least one other tested transgene pair, suggesting that RpS21 may, to some degree, act specifically to regulate the Ras/MAPK pathway at specific signaling intensity levels. Overall, our findings highlight the ability of our approach to reveal new Ras/MAPK regulators that preferentially impact specific signaling outputs.Our above results suggested that it is Ras/MAPK signaling strength, and not codon manipulation specifically, that determine whether RpS21 heterozygosity impacts protein expression. To test this idea further, we generated an additional pair of transgenes with identical protein sequence but distinct codon usage. Specifically, we generated two GFP transgenes- one with GFP containing 100% common codons, and one where the same GFP had 50% synonymous substitutions of rare codons dispersed throughout the protein. Both transgenes were expressed under a Drosophila eye) but do so with the new angle of altering codon usage in a core signaling component to find signal strength-dependent regulators. We show here that changing codon usage in a signaling pathway component can be an effective strategy to find signal strength-dependent modifiers, as evidenced by our identification of 15 Df from a whole-genome screen that only modify the rough-eye phenotype driven by either a common or rare codon-enriched RasV12 transgene, but not both. From these efforts, we identify the RpS21 gene as a negative regulator of a weak or low-level Ras phenotype in the in vivo context of eye development. These findings are further supported by our finding that RpS21 reduction in other contexts also impacts (low) endogenous Ras signaling in the ovary, but not higher Ras signaling in S2 cells.Here, we revisit a well-proven strategy to identify Ras/MAPK modifiers  and subsequently generated by Gene Synthesis . A cDNA clone  was used as a template to generate the non-altered Ras85D sequence. To generate RasV12Native, the QuikChange II Site-Directed Mutagenesis Kit (Agilent) was used to change codon 12 in Ras85D from GGA (glycine) to GTA . Subsequently, primers (sequences available upon request) were designed to amplify Ras sequences and the Invitrogen Gateway BP Clonase II Enzyme Mix (ThermoFisher Scientific) was used to insert these sequences into the Gateway entry vector pDONOR221 (ThermoFisher Scientific). Subsequently, the Invitrogen LR Clonase Enzyme Mix (ThermoFisher Scientific) was used to insert the RasWT, and RasV12 Native, Common, and Rare sequences into the Gateway destination vector pBID-UASC-FG (Addgene Plasmid #3520 [Ras plasmids were prepared with a ZymoPURE II Plasmid Midiprep Kit (Zymo Research) and sent to Model System Injections  for injection into attP40 (2L) flies. GFP sequences were cloned into a pBID plasmid (modified from Addgene Plasmid #3520), and DNA and transgenic flies were prepared as for Ras transgenes. For cell culture, RasV12Common and RasV12Rare transgenes were cloned into pMKInt-Hyg vectors, which were sequenced to confirm the correct sequence.Codon-altered exon sequences for id #3520 ), which http://FlyBase.org) describes full genotypes for all stocks used in this study. See S1 Table for Df stock information. All other stocks were the following genotypes (Bloomington Drosophila Stock Center numbers in parentheses when available): ksrS-627/TM3,Sb (#5683), aopyan-XE18/CyO (#8777), betaggt-IS-2554 (#5681), RpS2103575/CyO (#11339), hsflp;; UAS-GFP, tubulin-FRT-STOP-FRT-Gal4 [UAS-FLAG-Ras, UAS-FLAG-RasV12Native, UAS-FLAG-RasV12Common, and UAS-FLAG-RasV12Rare, ubi-GFP Rare, ubi-GFP Common.All flies were raised at 25°C on standard media unless noted otherwise . FlyBase . The scoring system was as follows : Mild = 1, no discoloration or necrotic tissue; Moderate = 3, discoloration and no necrotic tissue; Severe = 5, discoloration and necrotic tissue . Severity scores for each genotype was calculated as follows: (#Mildx1+#Moderatex3+#Severex5)/Total # of flies. To determine if heterozygosity for a subset of genes altered the rough eye phenotype the following two genotypes for each deficiency (Df) were compared: Ras transgene only and Ras transgene + Df . Then, we calculated a fold change score for both RasV12Common and RasV12Rare for each deficiency: Ras transgene + deficiency/Ras transgene. We note that none of the Df animals on their own had detectable eye phenotypes. For the primary screen, the fold change score was defined as follows: enhancer (fold change ≥1.35 or 5X less flies eclosed); suppressor (fold change ≤0.65 or 5X more flies eclosed). For the secondary screen, the fold change score was defined as follows: enhancer (fold change ≥1.95 or 5X more flies eclosed); suppressor (fold change ≤0.50 or 5X less flies eclosed). The final phenotype for a deficiency was defined as follows: not a modifier (neither RasV12Common or RasV12Rare + Df were modified); enhancer (both RasV12Common and RasV12Rare + Df were enhanced); suppressor (both RasV12Common and RasV12Rare + Df were enhanced); differential (only RasV12Common or RasV12Rare + Df were modified). We note that overall eye size was relatively unaffected by different Ras transgenes. Images of fly eyes were obtained using a Leica MZ10F microscope with a PlanApo 1.6X objective, Pixel Shift Camera DMC6200, and LASX software.To examine eye phenotypes and associated animal survival in such experiments, the Figs S5 samples were processed in Laemmli buffer and then boiled for 5min. Samples were separated by 12% sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gels and transferred to an Odyssey nitrocellulose membrane (LI-COR Biosciences) for immunoblotting. The following antibodies were used: anti-FLAG M2 , anti-α-tubulin , rabbit anti-GFP , IRDye 800CW , and Alexa Fluor 680 goat anti-mouse IgG (H+L) . Signal was detected using LI-COR Odyssey CLx and analyzed using Image Studio (LI-COR Biosciences). For all other immunoblots, samples were processed in RIPA buffer containing 1% IGEPAL, 50mM NaCl, 2mM EDTA, 100mM Tris-HCl, pH 8.0, 0.1% Glycerol, 50 mM Naf, 10mM Na3VO4, and protease inhibitors (Roche). Drosophila heads and ovaries were collected and transferred to cold lyses to be homogenized with a pellet pestle. Lysates were incubated at 4°C for 30 min on end-to-end rotator and then centrifuged at 21,000 x g for 10 min. The supernatant was transferred to a new tube. Total protein was quantified using a BCA kit (Bio-Rad) and 10 to 50 micrograms of protein was used for separation on either 12.5% or 15% gradient SDS-PAGE gels. Proteins on SDS gels were transferred onto polyvinylidene difluoride membranes. These membranes were probed with anti-Flag , anti-β-actin , anti-p-MEK1/2 , anti-MEK1/2 , anti-p-ERK1/2 , anti-ERK1/2 , and anti-RpS21  primary antibodies in blocking buffer containing 5% milk goat anti-mouse IgG (H+L) HRP  or goat anti-rabbit IgG (H+L) HRP . Immunoblots were visualized using Clarity Max ECL Western Blotting Detection Reagent (Bio-Rad) followed by exposure to digital acquisition using Chemi Doc Imager (Bio-Rad). For all blots, the contrast and/or brightness were altered equally across the entire image and then images were cropped for displaying as figures. Flag band intensity was measured using Image Lab v6.0.1 software and then each band was normalized to the lowest intensity band. The active Ras detection kit  was used to detect GTP-bound RasV12, both rare and common.All protein samples were prepared by homogenizing tissue on ice. For Ras constructs were designed against an identical region containing the 3xFLAG sequence shared by both RasV12Common and RasV12Rare transcripts. Primers were designed against Drosophila Beta Tubulin 56D as a reference gene. Ras qPCR FW primer: TGGACTACAAAGACCATGACGGT, Ras qPCR RV primer: ACTTGTATACCGGTGCTTGTCAT, Tubulin qPCR FW primer: GGACGAGACCTACTGCATCG, Tubulin qPCR RV primer: GGTCACCGTATGTGGGTGTC.Animals were aged 3–7 days at 25°C on standard fly medium. RNA was extracted from adult fly heads using TRIzol™ reagent  according to the manufacturer’s protocol (10–40 heads per sample in 500ul TRIzol™ reagent). Purified RNA was resuspended in molecular grade water. RNA was DNase treated with DNase I at room temp for 15 minutes, then the reaction was terminated by adding 25mM EDTA and incubating at 65°C for 10 minutes. DNase efficiency was confirmed using a positive control. DNase treated RNA was reverse transcribed into cDNA using iScript cDNA synthesis kit  according to the manufacturer’s protocol. Subsequent cDNA was treated with RNase H prior to use in qPCR reactions. The concentration of the RNA was quantified on a NanoDrop spectrophotometer and samples were diluted with molecular grade water to match the concentration of the lowest concentration sample. Luna Universal qPCR Master Mix (NEB #M3003) was used to run the qPCR reaction according to the manufacturer’s specifications. Primers for the detection of Drosophila medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin- L Glutamine (Invitrogen) at 25°C. FBS was heated for 60 minutes in 58°C and then cooled down before being added to the medium. These cells were confirmed to be free of mycoplasma infection, as measured by the Duke Cell Culture Facility using MycoAlert PLUS test (Lonza). S2 and KC cell lines were stably transduced with the pMKInt-Hyg vector encoding RasV12Common and RasV12Rare cDNAs using 1000 ng of DNA in 6 well plates per manufacturer instructions . The following day, Schneider’s media was changed, and cells were seeded in a coated culture dish (100x20 mm). Four days later, cells were passaged with fresh Schneider’s medium and 200 μg/ ml hygromycin (Invitrogen) was added. The stably transfected cells were selected within a month growing in media containing hygromycin. Three days prior to any experiment, these cells were grown in media without hygromycin. Four million S2 cells that were stably transduced with RasV12Common and RasV12Rare plasmids were seeded into coated tissue culture dishes  with 2 ml of Schneider’s media (without FBS). Sixty micrograms of RpS21 dsRNA were added on top of these cells. One hour later, two ml Schneider’s media containing 20% FBS were added on top of 2 ml Schneider’s media without FBS resulting in medium with 10% FBS concentration in total media of this culture. Within 16–24 hours after RNAi treatment, expression of RasV12Common and RasV12Rare transgenes were induced by CuSO4 for another 12 hours. Finally, these cells were collected 30–36 hours after dsRNA treatment.KC and S2 cell lines were obtained from Bloomington (Indiana University DGRC Bloomington) and as a gift from Dr. David MacAlpine (Duke University) respectively. These cells were cultured in Schneider’s RpS21 dsRNA production using the forward primer “TAATACGACTCACTATAGGGTTACTGACCAGCCGATACCC” and reverse primer “TAATACGACTCACTATAGGGCCACGCTTAGAAGTTCCTGC”. Next, 500 ng of RpS21 PCR template was used for an in vitro production of dsRNA as instructed in the MEGAscrip T7 transcription kit (ThermoFisher). The dsRNA solution was cleared using MegaClear kit (ThermoFisher). Finally, the concentration of RpS21 dsRNA was measured and stored in -80°C for future use.S2 cell DNA was used to produce a PCR template for http://genomes.urv.es/CAIcal [Drosophila Stock Center website. Deficiencies were then uploaded as a custom BED track to the UCSC Genome Browser (Reference Assembly ID: dm6). Genes overlapping the deficiency coordinates were then extracted using BEDtools for additional analysis [Ras modifiers if any of the deficiency covered genes known as Drosophila Ras85D genetic interactors . Enhancers and suppressor deficiencies were analyzed using the same metric against known Ras85D interactors of the same respective modifier type. Statistical analysis (chi-square) was performed using Graphpad Prism v8.1. FlyBase Gene Group Enrichment analysis was performed by comparing deficiency covered genes with pre-defined FlyBase Gene Groups. Analysis and statistical tests were performed in R using Gene Overlap package (https://rdrr.io/bioc/GeneOverlap/) and results are reported as adjusted p-values , sequences were entered at the CAIcal web-server  Alignments of Ras transgenes. Nucleotide changes highlighted for RasV12Common (red) and RasV12Rare (blue). Table with overall CAI score and GC content for Ras transgenes. (b) Codon Adaptation Index (CAI) plot. Transparent circles, squares, and triangles are individual CAIs per codon. Solid lines represent a best-fit line of individual points for each transgene. (c) Amino acid alignment of endogenous Drosophila Ras85D with human KRASA and KRASB, over a region of sequence divergence between KRASA and KRASB. The percent identity is noted. (d) Nucleic acid alignment of the four transgenes used in this study with human KRASB. (e) Nucleic acid phylogenetic tree of human KRASB and the four transgenes used in this study, with the percent identity of each gene/transgene to Drosophila RasWTNative indicated.(TIF)Click here for additional data file.S2 Fig(a) Representative image of adult eye from animal expressing sevGal4>RasWT(b) Immunoblot detection of transgenic RasV12 protein (with an anti-FLAG antibody) and αTubulin as a loading control from lysates derived from the head of flies with the indicated versions of transgenic RasV12. (c) Quantification of protein levels at 25°C for blot in S1B Fig. a.u. = arbitrary units. Data represent mean ± SEM, 3 replicates, Tukey’s multiple comparisons test. (d) Biological replicate of serial dilution of RasV12 common versus rare. 10, 20, and 30 ug of lysates derived from the heads of flies expressing the indicated versions of transgenic RasV12 were immunoblotted with an anti-FLAG antibody, demonstrating differential expression of RasV12 common and rare. Bottom: quantification and protein loaded. (d) Immunoblot detection of transgenic RasV12 (with an anti-FLAG antibody), phosphorylated (p-) and total Mek and Erk, and actin as a loading control from lysates derived from (e) the head of flies with the indicated versions of transgenic RasV12 or (f) S2 and KC cells stably transduced with expression vectors expressing the indicated RasV12 transgenes. First lane is S2 cells without any transfection. (g) Levels of GTP-bound RasV12 common versus rare. GTP-bound Ras from lysates derived from S2 cells stably expressing RasV12 common versus rare (or no transgene as a control) were affinity captured with a Ras Binding Domain (RBD IP) and immunoblotted with an anti-FLAG antibody to detect the ectopic active portion of the expressed RasV12 protein. Whole cell lysates (WCL) were immunoblotted with an anti-FLAG antibody to detect total ectopic RasV12 protein and Actin as a loading control. One representative blot from multiple replicates is shown.(TIF)Click here for additional data file.S3 Fig(a) Quantification of eye severity scores for Ras transgenes that are also heterozygous for known Ras modifiers. Data represent mean ± SEM, multiple replicates (using Dennett’s multiple comparison test). (b) The average number of flies eclosed per experiment for Rare and Common transgenes in a known Ras modifier background.(TIF)Click here for additional data file.S4 Fig(a) Genome map of Df(2L)BSC692. RpS21 is highlighted in green. (b) Immunoblot detection of transgenic RasV12 (with an anti-FLAG antibody), and actin as a loading control from lysates derived from the head of flies with the indicated versions of transgenic RasV12 in either the wild-type (+/+) or mutant (RpS2103575/+) backgrounds, (c) Immunoblot detection of transgenic RasV12 (with an anti-FLAG antibody),phosphorylated (p-) and total Mek and/or Erk, RpS21, and actin as a loading control from lysates derived from S2 cells stably transduced with expression vectors expressing the indicated RasV12 transgenes in the absence (-) and presence (+) of RpS21 RNAi (Data represent two independent replicates.)(TIF)Click here for additional data file.S5 Fig(a) Alignments of GFP transgenes. GFP Common contains all common codons. Nucleotide changes to generate rare codons in GFP Rare are highlighted in (blue). Table with overall CAI score and GC content for GFP transgenes. (b) Codon Adaptation Index (CAI) plot. Triangles are individual CAIs per codon. Solid lines represent a best-fit line of individual points for each transgene.(TIF)Click here for additional data file.S1 TableSee text for details.(XLSX)Click here for additional data file.


Ostm1) gene and protein, essential for proper osteoclast maturation, and responsible when mutated for the most severe form of osteopetrosis in mice and humans.The maintenance of bone mass is a dynamic process that requires a strict balance between bone formation and resorption. Bone formation is controlled by osteoblasts, while osteoclasts are responsible for resorption of the bone matrix. The opposite functions of these cell types have to be tightly regulated not only during normal bone development, but also during adult life, to maintain serum calcium homeostasis and sustain bone integrity to prevent bone fractures. Disruption of the control of bone synthesis or resorption can lead to an over accumulation of bone tissue in osteopetrosis or conversely to a net depletion of the bone mass in osteoporosis. Moreover, high levels of bone resorption with focal bone formation can cause Paget’s disease. Here, we summarize the steps toward isolation and characterization of the osteopetrosis associated trans-membrane protein 1 ( Osteoclasts derive from hematopoietic stem cells that are shared with early myeloid lineage precursors. Differentiation of osteoclast precursors is dependent on mature osteoblasts that produce macrophage colony-stimulating factor (M-CSF), receptor activator of NF-κB Ligand (RANKL), and osteoprotegerin (OPG) a soluble decoy receptor of RANKL ,2,3,4,5.The biochemical characterization of osteoclasts have been hampered by the fact that these giant cells are tightly attached to the bone matrix and are therefore difficult to isolate. Moreover, as these cells are terminally differentiated and non-proliferative, a large number of cells have to be isolated at once. However, despite these impediments, osteoclast specific markers have been defined and novel efficient tools have been developed to analyze osteoclast biology ex vivo and in vivo .+/V-ATPase and chloride exchanger ClC-7 in acidification of the extracellular resorbing lacunae to ensure bone matrix demineralization [When osteoclasts are activated, a resorption cycle is induced causing several proteins to be relocalized along with cytoskeletal rearrangement. Active osteoclasts are polarized and show two cellular histo-morphologic characteristics: an actin ring and a ruffled border. The actin ring, devoided of organelles, is enriched in dynamic and adhesive projections of the cell membrane called podosomes and in αVβ3 integrins that allow spreading and tight attachment to the bone surface ,10,11. Tlization ,17,18. Ilization , and catlization  whereas lization . Importalization . Furtherlization ,24,25,26infantile-malignant autosomal recessive (ARO) which is fatal within the first few years of life;intermediate recessive (IRO) which appears during the first decade of life but does not mediate a malignant response;autosomal dominant (ADO), with full-life expectancy but with major bone malformations.Defective osteoclast differentiation or generation of inefficient osteoclasts leads to the severe bone pathology called osteopetrosis, a heterogenous inherited disease of bone metabolism ,28. ThisEach form of the disease is characterized by a reduced bone marrow compartment leading to hematopoietic defects including anemia and high susceptibility to infections ,36. Chargl) mouse mutant was described by Gruneberg [gl/gl mice display a severe growth delay and a grey-coat color on an agouti background due to pheomelanin granule clumping [The spontaneous osteopetrotic grey-lethal  that give rise to osteoclasts and consistent with an increase of splenic CD11b+/Lys6-G+ monocytic cell subpopulation. In addition to this myeloid defect, deregulation of lymphoid lineages in gl/gl mice resulted in a reduction of B cell populations and altered T cell distribution with thymus hypo-cellularity [gl gene in lymphoid cell lineage.Restoration of the capacity to resorb bone matrix following normal spleen and/or bone marrow cells transplantation in c defect . It is nc defect . Our chalularity . This regl/gl bone tissue demonstrated the presence of numerous mature multinucleated osteoclasts, suggesting an intrinsic osteoclast defect that excluded cell differentiation impairment due to environmental factors. Consistent with this, ultra-structural analysis of bone sections showed that gl/gl osteoclasts are in close contact to the bone matrix but display an underdeveloped ruffled border essential for proper bone matrix resorption [gl/gl osteoclasts in culture demonstrated normal spreading through formation of an intact actin ring but these cells were unable to resorb bone matrix. The presence of an inactive mature osteoclast population in these mice classified the gl/gl phenotype as an ‘osteoclast-rich’ osteopetrosis [Importantly, in situ histological characterization of sorption . Accordipetrosis .gl mutation that most closely resembles human recessive osteopetrosis. By generating—for the first time—two backcross panels penetrant for the gl mutation, we have produced a genetic map and reduced the genetic interval on murine chromosome 10 that included the gl locus from 5 cM to ~1 cM [gl candidate region to a ~1000 kb genomic interval. This interval was then covered with a contig of 17 overlapping bacterial artificial chromosomes (BAC) and novel polymorphic markers narrowed our candidate interval to ~500 kb. BAC transgenic lines were produced and full functional rescue obtained with one BAC clone in transgenic homozygous gl/gl mice defined a 180kb genomic candidate segment for the localization of the gl locus. BAC sequencing and transcription sequence analysis defined a single gene, called Ostm1, which encodes a unique 3kb transcript highly expressed in osteoclasts and undetectable in homozygous gl/gl animals [Ostm1 (omOstm1) was detected in an N-ethyl-N-nitrosourea (ENU) screen and results in a mild osteopetrotic phenotype but the mutation still needs to be defined [We have successfully used a positional cloning strategy to isolate and characterize the gene responsible for the mouse osteopetrotic to ~1 cM . During to ~1 cM . Additio animals . Of noteOstm1 is widely expressed and detectable in embryonic hematopoietic, skeletal, and brain tissues which is maintained after birth along with an appearance in gut, kidney, and skin. Subsequently, full-length cDNA was isolated and we established that the Ostm1 gene is composed of six exons and five introns. In contrast to its wild-type counterpart, gl genomic DNA has a ~7.5 kb deletion. This deletion covers most of the promoter, the first exon, and a large portion of the first intron leading to a null allele [Ostm1 in hematopoietic cells of transgenic mice with the regulatory sequences of the transcriptional factor gene PU.1 (PU.1-Ostm1) resulted in full rescue of osteopetrosis and hematopoietic defects [Ostm1 is the gene responsible for the gl mutation. Subsequently, we isolated the human OSTM1 gene and a database search identified homologs only in metazoans. Interestingly, genomic sequence analysis of 19 osteopetrotic patients led us to characterize the first human OSTM1 mutation associated with the disease that results in exon 5 skipping. This result was confirmed at the RNA level and allowed us to design and apply the first prenatal diagnostic test for carriers [OSTM1 mutations with severe osteopetrosis also displayed neurological disorders [l allele . Importa defects . This prcarriers ,51. Addiisorders ,53 suggeisorders ,55.gl/gl hematopoietic tissue identified the Inositol polyphosphate 4-phosphatase type II (Inpp4b) transcript as constantly downregulated. First, we isolated and characterized the Inpp4b gene in the mouse [Inpp4b in the mouse was induced and we demonstrated that Inpp4b is a negative regulator of osteoclast differentiation ex vivo. These mice consistently develop an osteoporotic phenotype in vivo, linking lipid metabolism to a specific bone phenotype. In humans, we showed that specific INPP4B variants were associated with variable bone mineral density and established INPP4B as a susceptibility locus to osteoporosis in pre-menopausal women [Ostm1 and Inpp4b remains to be elucidated, these results indicate that additional genomic loci can be deregulated due to the absence of Ostm1 expression and may give rise to the discovery of novel regulators of bone mineral density in mouse and human.In parallel, gene expression profile analyses of he mouse . Second,al women . NeverthOstm1 gene encodes a 338-amino acid protein while the 334 amino acid human OSTM1 protein is 83% homologous to the mouse protein. Our structural analysis defined a signal peptide and a unique trans-membrane domain that classified Ostm1 as a type I trans-membrane protein where the majority of Ostm1 is luminal with a short cytosolic 30 amino acid C-terminus  screen using a tagged version of the C-terminus of Ostm1  and iden−-H+ exchanger, as a partner of Ostm1 [Directly related to osteoclast biology we, and others, have also detected the protein ClC-7, a Clof Ostm1 . The Ostof Ostm1 ,65. Howeof Ostm1 . This coof Ostm1 .Ostm1lox allele to induce conditional ablation (cKO) of Ostm1 protein in any tissue. As the first human mutation, we described results from OSTM1 exon 5 skipping, loxP sites flanking exon 5 were introduced in the mouse locus to mimic the human mutation [gl phenotype by crossing Ostm1lox/lox mice with systemic deletion via Meox-Cre+ deleter transgenic line [Ostm1lox/lox Meox-Cre+ progenies develop severe osteopetrosis and die ~3 weeks after birth. Thus, we generated the first engineered Ostm1 cKO mouse model.To analyze the role of Ostm1 in a cell specific manner, we generated an mutation . We valinic line . AccordiOstm1 was induced in mature osteoclasts with the Cathepsin K (Ctsk-Cre) deleter transgenic line [Ostm1Δexon5 Ctsk-Cre+ homozygous mice. An in-frame exon 5 deletion was subsequently confirmed by sequencing and the recombination level in mature Ostm1lox/lox Cre+ osteoclasts approached 100%. Ostm1lox/lox Ctsk-Cre+ mice develop severe osteopetrosis similar to the gl/gl phenotype with a short lifespan of ~3 weeks [Ostm1Δexon5/+ Ctsk-Cre+ mice that display a normal phenotype, excluding a dominant negative effect for the truncated/secreted Ostm1 protein as proposed in cell culture systems [OSTM1 patients that are asymptomatic.Subsequently, cKO of nic line  to gener~3 weeks . We also systems . Our resOstm1 loss of function, we first showed that conditional ablation of Ostm1 in osteoclasts does not affect osteoclast differentiation but results in formation of numerous oversized multinucleated osteoclasts in vivo and ex vivo [DC-Stamp), osteoclast stimulatory trans-membrane protein , and the d2 isoform of vacuolar ATPase V0 domain (Atp6v0d2) is essential in this mechanism [DC-Stamp null mice that exhibit formation of only mononucleated pre-osteoclasts less capable to resorbing bone matrix, whereas transgenic mice overexpressing DC-Stamp accumulate osteoclasts of greater size with accelerated fusion [Atp6v0d2 gene led to reduced osteoclast fusion and defective bone resorption [Ostm1, a significant increase in transcription upregulation of these fusogenic genes was quantified in the oversized multinucleated cKO osteoclasts. This result was further substantiated with the upregulated expression and nuclear relocalization of the transcription factor Nfatc1, the upstream master regulator that controls expression of these target genes [Analogous to systemic  ex vivo  similar  ex vivo . Normall ex vivo ,72,73. Eechanism ,75,76. Techanism . The rold fusion . Similaret genes . Togetheet genes . Ostm1lox/lox Ctsk-Cre+ mice were larger in size and were able to form a peripheral actin ring and podosomes, critical for osteoclast tight attachment to bone matrix. However, these cKO cells are defective in bone resorption similar to osteoclasts with complete loss of Ostm1 in gl/gl mice. In normal conditions, upon bone matrix interaction, polarization of multinucleated osteoclasts induces cytoskeletal rearrangements and formation of a ruffled border essential for proper bone resorption [+/V-ATPase) and chloride transporter ClC-7 that secrete hydrogen and chloride ions into the lacunae [+/V-ATPase in mouse and humans was characterized by mutations in the a3 , Atp6v1c1 and Atp6ap1 (Ac45) enzyme subunits that result in non-functional osteoclasts [Secondly, we demonstrated in vitro that cKO osteoclasts isolated from sorption . This stsorption ,20,80. Isorption ,82 and d lacunae . The impeoclasts ,85,86,87eoclasts ,88.Ostm1 cKO osteoclasts, we also demonstrated that the acidic luminal pH of the secretory lysosomes, essential for their function, was not altered [gl/gl fibroblasts and neurons [In  altered  as descr neurons . However neurons . This re neurons   and Ctsk genes expression levels and proteins release. We showed that transcription levels of Acp5 and Ctsk were significantly enhanced in Ostm1 cKO osteoclasts. This response was consistent with concomitant upregulation of the Nfatc1 transcriptional factor expression since both Acp5 and Ctsk gene promotors are targets of Nfatc1. We then determined if the loss of Ostm1 can affect osteoclast protease release through biochemical quantification of extracellular levels of Trap and Ctsk enzymes. In Ostm1 cKO osteoclast cultures, Trap release was strongly reduced whereas the secreted Ctsk protein was undetectable. Impaired release of these proteases confirms that Ostm1 plays a role in osteoclast secretory lysosome trafficking and possibly in exocytosis  pathway and downstream targets [Ostm1 in the central nervous system (CNS) showed some similarities with known lysosomal storage diseases like Parkinson’s and Alzheimer’s [OSTM1 patients further supporting an essential primary role of Ostm1 in the CNS, independent of the hematopoietic lineage [Our cellular analysis in these mice unraveled a marked accumulation of autophagosomes in neurons from the cortex and hippocampus, indicative of an impaired autophagy mechanism. This phenotype results in axonal swelling consistent with a trafficking defect . In this targets . These pheimer’s ,93,94. M lineage .OSTM1 mutations are responsible for the most severe form of infantile type 3 autosomal recessive osteopetrosis with neuropathy (OMIM no. 259720) [OSTM1 micro-deletions defined a new class of mutations to be considered in diagnostic screening [ 259720) ,95,96. P 259720) ,53,97,98creening . In paracreening . Until now, allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for ARO but the success rate is not optimal with engraftment being a limiting factor and overall outcomes remain disappointing ,103,104.−/−)oc  mice . Alterna/−) mice . As vali/−) mice  and coulablished . Despitepatients . HoweverOstm1 locus responsible for the most severe form of osteopetrosis in mice and humans. All of these studies were made possible through generation of mouse models as well as molecular and cellular protocols to analyze osteoclast phenotypes. In osteoclasts, Ostm1 is a negative modulator of cell multi-nucleation, an essential step toward cell maturation. In osteoclast activation, Ostm1 along with specific partners is a positive regulator of intracellular trafficking of secretory lysosomes responsible for ruffled border formation, extracellular acidification, and bone matrix degradation. Therefore, Ostm1 expression in mature osteoclasts is absolutely required to prevent osteopetrosis. For OSTM1 patients, major progress has been made by the design of prenatal screens in carrier families, however more studies are needed to develop efficient curative treatments since these patients can frequently relapse independently from the hematopoietic cell lineage itself. The characterization of the molecular mechanisms of Ostm1 was of fundamental importance for our understanding of osteoclast biology, but also of high clinical relevance for bone diseases like osteopetrosis. Further studies in mice are still required, including discovery of additional Ostm1 partners to get insights in the complex bone cell crosstalk responsible for maintenance of bone matrix homeostasis. The osteoclast remains a fascinating cell to study and a better knowledge of its biology and multiple cellular interactions will characterize novel therapeutic targets for major bone diseases.Our successful quest to understand the mouse grey-lethal osteopetrotic mutation was a long journey but it has allowed us to characterize the previously unknown