Molecular Study from the Signaling Pathways of Four Potential asthma triggers: AKT1, MAPK13, STAT1, and TLR4
Abstract
1. Introduction
2. Results
2.1. Subjects
2.2. The Four Potential Gene Triggers Show Different Gene and Protein Expression in Asthma Patients than in the Healthy Subjects
2.3. A Deeper Analysis Showed Differences in the Results Obtained for Each Clinical Group When Patients Were Segregated by Severity
2.4. The Expression of the Four Potential Triggers Showed a Good Gene Correlation Between Them, but Worse at a Protein Level, with Differences Among Clinical Groups
2.5. The Four Potential Triggers Showed Different Correlation with Lung Functional Parameters Depending of the Group Studied
2.6. Analysis of the Signaling Pathways of the Triggers Proteins Show a Significant Gene Expression Decrease in Asthma Groups Compared to HC Subjects
2.7. NA Patients Have Lower Gene Expression than AA Patients on the Common Genes to Both Signaling Pathways
3. Discussion
4. Materials and Methods
4.1. Study Design: Subjects
4.2. Isolation of Peripheral Blood Mononuclear Cells, RNA, and Protein Extraction
4.3. Gene Selection and Differential Expression Analysis by RT-qPCR
4.4. Differential Protein Expression Analysis by Western Blot
4.5. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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HC | AA | NA | |
---|---|---|---|
N | 15 | 45 | 17 |
Age (y.o), mean ± SD | 32.87 ± 10.8 | 48.02 ± 12 ## | 54.88 ± 11 ## |
Gender, N (%) | |||
Male | 7 (46.7) | 12 (26.7) | 7 (41.2) |
Female | 8 (53.3) | 33 (73.3) | 10 (58.8) |
Smoking habit, N (%) | |||
Smoker | 5 (11.1) | 2 (11.8) | |
Ex- Smoker | 13 (28.9) | 9 (52.9) | |
No Smoker | 25 (55.6) | 6 (35.3) | |
ND | 15 (100) | 2 (4.4) | 0 (0) |
BMI (Kg/m2), mean ± SD | 23.6 ± 1.3 | 25.87 ± 4.5 | 27.5 ± 4.8 * |
Asthma severity, N (%) | |||
Severe | - | 17 (37.8) | 4 (23.5) |
Moderate | - | 14 (31.1) | 8 (47) |
Mild | - | 14 (31.1) | 5 (29.5) |
Spirometry, mean ± SD | |||
FEV1 (%) | 100.82 ± 7.9 | 87.74 ± 20 | 96.89 ± 21.8 |
FVC (%) | 104 ± 6.9 | 98.88 ± 18.7 | 110.1 ± 20.6 |
FEV1/FVC | 82.53 ± 2.2 | 77.09 ± 9.8 | 81.78 ± 14.4 |
FeNO (ppb), mean ± SD | 11.1 ± 5.9 | 38.4 ± 30.7 ** | 32.38 ± 27 |
Total IgE (IU/mL), mean ± SD | 63.55 ± 94.7 | 436.71 ± 493.1 #,† | 159.95 ± 173.2 * |
Blood cells | |||
Eosinophils (cells/mm3), mean ± SD | 280 ± 168.7 | 324.67 ± 206.1 | 424.8 ± 205 |
Neutrophils (cells/mm3), mean ± SD | ND | 3823 ± 1574.6 | 4438.33 ± 1172.6 |
Sputum cells | |||
Eosinophils (%), mean ± SD | ND | 9.6 ± 17.4 | 16 ± 27.9 |
≥3% eosinophils, N (%) | ND | 10 (22.2) | 5 (29.4) |
Neutrophils (%), mean ± SD | ND | 29.4 ± 24 | 40 ± 30.4 |
≥70% neutrophils, N (%) | ND | 4 (8.8) | 3 (17.6) |
Cellular profile, N (%) | |||
Eosinophilic | ND | 9 (20) | 5 (29.4) |
Neutrophilic | ND | 2 (4.4) | 3 (17.7) |
Mixed | ND | 1 (2.2) | 0 (0) |
Paucigranulocyte | ND | 13 (28.9) | 4 (23.5) |
ND | ND | 19 (42.2) | 5 (29.4) |
Treatment, N (%) | |||
Inhaled Corticosteroids (ICS) | - | 43 (95.5) | 17 (100) |
Systemic Corticosteroids | - | 1 (2.2) | 2 (11.8) |
Long-term β2-Agonists (LABA) | - | 39 (86.7) | 17 (100) |
Short-term anticholinergics | - | 1 (2.2) | 1 (5.9) |
Long-term anticholinergics | - | 9 (2) | 2 (11.8) |
Leukotriens receptor agonists | - | 12 (26.7) | 3 (17.7) |
Short-term bronchodilators | - | 18 (40) | 7 (41.2) |
Specific immunotherapy | - | 12 (26.7) | 0 (0) |
Biologics (Omaluzimab, Mepaluzimab) | - | 14 (31.1), 4 (8.8) | 2 (11.8), 1(5.9) |
| ||||
AKT1 | MAPK13 | STAT1 | TLR4 | |
AKT1 | Rs = 0.97; p < 0.001 | Rs = 0.73; p = 0.002 | Rs = 0.60; p = 0.02 | |
MAPK13 | Rs = 0.82; p = 0.004 | Rs = 0.69; p = 0.005 | Rs = 0.64; p = 0.01 | |
STAT1 | Rs = 0.51; p = ns | Rs = 0.57; p = ns | Rs = 0.55; p = 0.04 | |
TLR4 | Rs = 0.89; p = 0.001 | Rs = −0.19; p = ns | Rs = 0.74; p = 0.02 | |
| ||||
AKT1 | MAPK13 | STAT1 | TLR4 | |
AKT1 | Rs = 0.86; p < 0.001 | Rs = 0.92; p < 0.001 | Rs = 0.71; p < 0.001 | |
MAPK13 | Rs = −0.41; p = ns | Rs = 0.83; p < 0.001 | Rs = 0.60; p < 0.001 | |
STAT1 | Rs = 0.10; p = ns | Rs = 0.74; p = 0.01 | Rs = 0.75; p < 0.001 | |
TLR4 | Rs = −0.47; p = ns | Rs = 0.59; p = ns | Rs = 0.84; p < 0.001 | |
| ||||
AKT1 | MAPK13 | STAT1 | TLR4 | |
AKT1 | Rs = 0.92; p < 0.001 | Rs = 0.94; p < 0.001 | Rs = 0.53; p = 0.04 | |
MAPK13 | Rs = −0.53; p = ns | Rs = 0.83; p < 0.001 | Rs = 0.53; p = 0.04 | |
STAT1 | Rs = −0.35; p = ns | Rs = −0.36; p = ns | Rs = 0.53; p = 0.04 | |
TLR4 | Rs = 0.24; p = ns | Rs = −0.62; p = 0.05 | Rs = 0.55; p = ns |
| |||||
FEV1 | FVC | FEV/FVC | PBE | ||
AKT1 | Gene | Rs = −0.28; p = ns | Rs = −0.30; p = ns | Rs = −0.15; p = ns | Rs = 0.28; p = ns |
Protein | Rs = −0.59; p = ns | Rs = −0.62; p = ns | Rs = −0.16; p = ns | Rs = −0.16; p = ns | |
MAPK13 | Gene | Rs = −0.39; p = 0.009 | Rs = −0.36; p = 0.02 | Rs = −0.13; p = ns | Rs = 0.07; p = ns |
Protein | Rs = 0.27; p = ns | Rs = 0.36; p = ns | Rs = −0.38; p = ns | Rs = −0.19; p = ns | |
STAT1 | Gene | Rs = −0.41; p = 0.006 | Rs = −0.41; p = 0.005 | Rs = −0.25; p = ns | Rs = 0.30; p = 0.04 |
Protein | Rs = −0.63; p = 0.01 | Rs = −0.41; p = ns | Rs = −0.14; p = ns | Rs = 0.15; p = ns | |
TLR4 | Gene | Rs = −0.19; p = ns | Rs = −0.23; p = ns | Rs = −0.41; p = 0.009 | Rs = 0.15; p = ns |
Protein | Rs = −0.62; p = 0.02 | Rs = −0.62; p = 0.01 | Rs = 0.24; p = ns | Rs = 0.15; p = ns | |
| |||||
FEV1 | FVC | FEV/FVC | PBE | ||
AKT1 | Gene | Rs = 0.24; p = ns | Rs = 0.45; p = ns | Rs = 0.39; p = ns | Rs = 0.06; p = ns |
Protein | Rs = −0.08; p = ns | Rs = 0.53; p = ns | Rs = −0.25; p = ns | Rs = 0.02; p = ns | |
MAPK13 | Gene | Rs = 0.17; p = ns | Rs = 0.36; p = ns | Rs = 0.49; p = 0.04 | Rs = 0.06; p = ns |
Protein | Rs = 0.04; p = ns | Rs = 0.57; p = ns | Rs = 0.19; p = ns | Rs = −0.24; p = ns | |
STAT1 | Gene | Rs = 0.25; p = ns | Rs = 0.46; p = ns | Rs = 0.36; p = ns | Rs = 0.09; p = ns |
Protein | Rs = 0.43; p = ns | Rs = 0.58; p = 0.04 | Rs = −0.06; p = ns | Rs = −0.56; p = 0.04 | |
TLR4 | Gene | Rs = −0.22; p = ns | Rs = 0.16; p = ns | Rs = −0.15; p = ns | Rs = −0.11; p = ns |
Protein | Rs = 0.65; p = 0.03 | Rs = 0.85; p = 0.002 | Rs = 0.20; p = ns | Rs = −0.13; p = ns |
| ||||||||||
Gene | Gene Classification | Total AA | Severe AA | Moderate AA | Mild AA | |||||
RQ | Adj P | RQ | Adj P | RQ | Adj P | RQ | Adj P | |||
AKT1 pathway | AKT1 | Trigger | 0.177 | <0.001 | 0.109 | <0.001 | 0.154 | <0.001 | 0.354 | ns |
COL5A1 | Link Node | 0.177 | 0.004 | 0.067 | <0.001 | 0.203 | ns | 0.467 | ns | |
MMP9 | 0.041 | <0.001 | 0.027 | <0.001 | 0.033 | 0.006 | 0.082 | ns | ||
RELA | 0.165 | <0.001 | 0.088 | <0.001 | 0.154 | <0.001 | 0.379 | ns | ||
SMAD3 | 0.25 | <0.001 | 0.134 | <0.001 | 0.218 | <0.001 | 0.616 | ns | ||
TLR2 | 0.102 | <0.001 | 0.051 | <0.001 | 0.117 | <0.001 | 0.177 | ns | ||
MAPK13 pathway | MAPK13 | Trigger | 0.177 | <0.001 | 0.109 | <0.001 | 0.154 | <0.001 | 0.354 | ns |
IL12A | Link Node | 0.218 | 0.011 | 0.154 | <0.001 | 0.165 | 0.018 | 0.435 | ns | |
IL13RA1 | 0.117 | <0.001 | 0.072 | <0.001 | 0.125 | <0.001 | 0.177 | 0.029 | ||
NFATC1 | 0.144 | <0.001 | 0.067 | <0.001 | 0.117 | <0.001 | 0.5 | ns | ||
RELA | 0.165 | <0.001 | 0.088 | <0.001 | 0.154 | <0.001 | 0.379 | ns | ||
STAT1 pathway | STAT1 | Trigger | 0.117 | <0.001 | 0.063 | <0.001 | 0.109 | <0.001 | 0.287 | ns |
CCL5 | Link Node | 0.145 | <0.001 | 0.067 | <0.001 | 0.127 | <0.001 | 0.412 | ns | |
COL5A1 | 0.177 | 0.004 | 0.067 | <0.001 | 0.203 | ns | 0.467 | ns | ||
IL10 | 0.067 | <0.001 | 0.038 | <0.001 | 0.088 | 0.008 | 0.117 | ns | ||
IL12A | 0.218 | 0.011 | 0.154 | 0.001 | 0.165 | 0.018 | 0.435 | ns | ||
MMP9 | 0.041 | <0.001 | 0.027 | <0.001 | 0.033 | 0.006 | 0.082 | ns | ||
RELA | 0.165 | <0.001 | 0.088 | <0.001 | 0.154 | <0.001 | 0.379 | ns | ||
TLR4 pathway | TLR4 | Trigger | 0.072 | <0.001 | 0.038 | <0.001 | 0.072 | <0.001 | 0.144 | ns |
MMP9 | Link Node | 0.041 | <0.001 | 0.027 | <0.001 | 0.033 | 0.006 | 0.082 | ns | |
RELA | 0.165 | <0.001 | 0.088 | <0.001 | 0.154 | <0.001 | 0.379 | ns | ||
SMAD3 | 0.250 | <0.001 | 0.134 | <0.001 | 0.218 | <0.001 | 0.616 | ns | ||
Common | ALOX5 | Mechanistic | 0.102 | <0.001 | 0.044 | <0.001 | 0.088 | <0.001 | 0.287 | ns |
BAX | 0.233 | <0.001 | 0.154 | <0.001 | 0.203 | <0.001 | 0.5 | ns | ||
IL4R | 0.134 | <0.001 | 0.063 | <0.001 | 0.117 | <0.001 | 0.435 | ns | ||
TGFB1 | 0.180 | <0.001 | 0.11 | <0.001 | 0.157 | <0.001 | 0.337 | ns | ||
| ||||||||||
Gene | Gene classification | Total NA | Severe NA | Moderate NA | Mild NA | |||||
RQ | Adj P | RQ | Adj P | RQ | Adj P | RQ | Adj P | |||
AKT1 pathway | AKT1 | Trigger | 0.067 | <0.001 | 0.072 | 0.006 | 0.067 | <0.001 | 0.054 | <0.001 |
CDH1 | Link Node | 0.044 | <0.001 | 0.077 | ns | 0.038 | <0.001 | 0.036 | <0.001 | |
RELA | 0.062 | <0.001 | 0.054 | 0.002 | 0.072 | <0.001 | 0.058 | <0.001 | ||
SMAD3 | 0.125 | <0.001 | 0.117 | 0.002 | 0.134 | <0.001 | 0.109 | <0.001 | ||
TLR2 | 0.044 | <0.001 | 0.027 | 0.002 | 0.036 | <0.001 | 0.088 | 0.004 | ||
MAPK13 pathway | MAPK13 | Trigger | 0.036 | <0.001 | 0.022 | 0.004 | 0.033 | <0.001 | 0.058 | <0.001 |
CXCL8 | Link Node | 0.058 | ns | 0.054 | ns | 0.054 | ns | 0.072 | ns | |
NFATC1 | 0.067 | <0.001 | 0.063 | 0.002 | 0.067 | <0.001 | 0.077 | <0.001 | ||
RELA | 0.062 | <0.001 | 0.054 | 0.002 | 0.072 | <0.001 | 0.058 | <0.001 | ||
STAT1 pathway | STAT1 | Trigger | 0.051 | <0.001 | 0.044 | 0.002 | 0.063 | <0.001 | 0.047 | <0.001 |
CXCR3 | Link Node | 0.077 | <0.001 | 0.088 | 0.019 | 0.077 | <0.001 | 0.082 | 0.001 | |
RELA | 0.062 | <0.001 | 0.054 | 0.002 | 0.072 | <0.001 | 0.058 | <0.001 | ||
VCAN | 0.058 | <0.001 | 0.041 | 0.002 | 0.054 | <0.001 | 0.095 | <0.001 | ||
TLR4 pathway | TLR4 | Trigger | 0.036 | <0.001 | 0.022 | 0.004 | 0.033 | <0.001 | 0.058 | <0.001 |
CDH1 | Link Node | 0.044 | <0.001 | 0.077 | ns | 0.038 | <0.001 | 0.036 | <0.001 | |
RELA | 0.062 | <0.001 | 0.054 | 0.002 | 0.072 | <0.001 | 0.058 | <0.001 | ||
SMAD3 | 0.125 | <0.001 | 0.117 | 0.002 | 0.134 | <0.001 | 0.109 | <0.001 | ||
TLR5 | 0.051 | <0.001 | 0.102 | ns | 0.038 | <0.001 | 0.054 | 0.002 | ||
Common | ALOX5 | Mechanistic | 0.038 | <0.001 | 0.038 | 0.002 | 0.038 | <0.001 | 0.036 | <0.001 |
CCL5 | 0.058 | <0.001 | 0.051 | 0.002 | 0.063 | <0.001 | 0.054 | <0.001 | ||
PTGER2 | 0.067 | <0.001 | 0.036 | 0.002 | 0.077 | <0.001 | 0.077 | <0.001 |
Gene | Gene Classification | AA RQ | NA RQ | Adjusted P | |
---|---|---|---|---|---|
AKT1 Pathway | AKT1 | Trigger | 0.177 | 0.067 | 0.008 |
RELA | Link Node | 0.165 | 0.062 | <0.001 | |
SMAD3 | 0.250 | 0.125 | 0.021 | ||
TLR2 | 0.102 | 0.044 | 0.022 | ||
MAPK13 Pathway | MAPK13 | Trigger | 0.177 | 0.036 | 0.021 |
NFATC1 | Link Node | 0.144 | 0.067 | 0.028 | |
RELA | 0.165 | 0.062 | <0.001 | ||
STAT1 pathway | STAT1 | Trigger | 0.117 | 0.051 | 0.007 |
RELA | Link Node | 0.165 | 0.062 | <0.001 | |
TLR4 pathway | TLR4 | Trigger | 0.072 | 0.036 | ns |
RELA | Link Node | 0.165 | 0.062 | <0.001 | |
SMAD3 | 0.250 | 0.125 | 0.021 | ||
Commons | ALOX5 | Mechanistic | 0.102 | 0.038 | 0.011 |
Gene | Definition of the Gene According to Biology Systems [12] | Chromosome Location (GRCh38/hg38) | Primer Reference |
---|---|---|---|
AKT1 | Trigger | Chr.14: 104769349–104795743 | Hs00178289_m1 |
ALOX5 | Mechanistic in NA and AA | Chr.10: 45374166–45446121 | Hs01095330_m1 |
BAX | Mechanistic in AA | Chr.19: 48954825–48961798 | Hs00180269_m1 |
CCL5 | Mechanistic in NA, link node in AA | Chr.17: 35871491–35880373 | Hs00982282_m1 |
CCL11 | Mechanistic in NA | Chr.17: 34285668–34288180 | Hs00237013_m1 |
CCL17 | Link node in NA | Chr.16: 57396076–57416063 | Hs00171074_m1 |
CDH1 | Link node in NA | Chr.16: 68737290–68835542 | Hs01023895_m1 |
COL5A1 | Link node in AA | Chr.9: 134641790–134844843 | Hs00609088_m1 |
CXCR3 | Link node in NA | Chr.X: 71615913–71618517 | Hs01847760_s1 |
IL4R | Mechanistic in AA | Chr.16: 27313668–27364778 | Hs00166237_m1 |
IL8 (CXCL8) | Link node in NA | Chr.4: 73740506–73743716 | Hs00174103_m1 |
IL10 | Link node in AA | Chr.1: 206767603–206772494 | Hs00961622_m1 |
IL12A | Link node in AA | Chr.3: 159988836–159996019 | Hs01073447_m1 |
IL12B | Link node in AA | Chr.5: 159314783–159330473 | Hs01011518_m1 |
IL13RA1 | Link node in AA | Chr.X: 118726954–118794533 | Hs00609817_m1 |
IL17A | Mechanistic in NA and AA | Chr.6: 52186387–52190638 | Hs00174383_m1 |
MAPK13 | Trigger | Chr.6: 36130484–36144524 | Hs00559623_m1 |
MUC5B | Mechanistic in NA and AA | Chr.11: 1223065–1262176 | Hs00861595_m1 |
MMP9 | Link node in AA | Chr.20: 46008908–46016561 | Hs00957562_m1 |
NFATC1 | Link node in NA and AA | Chr.18: 79395772–79529323 | Hs00542678_m1 |
PTGER2 | Mechanistic in NA | Chr.14: 52314298–52328606 | Hs04183523_m1 |
RELA | Link node in NA and AA | Chr.11: 65653596–65662972 | Hs00153294_m1 |
SMAD3 | Link node in NA and AA | Chr.15: 67065698–67195195 | Hs00969210_m1 |
STAT1 | Trigger | Chr.2: 190969036–191014250 | Hs01013996_m1 |
TGFB1 | Mechanistic in AA | Chr.19: 41330531–41353933 | Hs00998133_m1 |
TLR2 | Link node in NA and AA | Chr.4: 153684080–153710643 | Hs00152932_m1 |
TLR4 | Trigger | Chr.9: 117704175–117717491 | Hs00152939_m1 |
TLR5 | Link node in NA | Chr.1: 223108401–223143282 | Hs01920773_s1 |
VCAN | Link node in NA | Chr.5: 83471674–83582303 | Hs00171642_m1 |
18S | Endogenous | - | Hs99999901_s1 |
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Cremades-Jimeno, L.; López-Ramos, M.; Fernández-Santamaría, R.; De Pedro, M.Á.; Mahillo, I.; Rosales-Ariza, C.; Olaguibel, J.M.; Pozo, V.d.; Caballero, M.L.; Luna-Porta, J.A.; et al. Molecular Study from the Signaling Pathways of Four Potential asthma triggers: AKT1, MAPK13, STAT1, and TLR4. Int. J. Mol. Sci. 2025, 26, 6240. https://doi.org/10.3390/ijms26136240
Cremades-Jimeno L, López-Ramos M, Fernández-Santamaría R, De Pedro MÁ, Mahillo I, Rosales-Ariza C, Olaguibel JM, Pozo Vd, Caballero ML, Luna-Porta JA, et al. Molecular Study from the Signaling Pathways of Four Potential asthma triggers: AKT1, MAPK13, STAT1, and TLR4. International Journal of Molecular Sciences. 2025; 26(13):6240. https://doi.org/10.3390/ijms26136240
Chicago/Turabian StyleCremades-Jimeno, Lucía, María López-Ramos, Rubén Fernández-Santamaría, María Ángeles De Pedro, Ignacio Mahillo, Cristina Rosales-Ariza, José María Olaguibel, Victoria del Pozo, María Luisa Caballero, Juan Alberto Luna-Porta, and et al. 2025. "Molecular Study from the Signaling Pathways of Four Potential asthma triggers: AKT1, MAPK13, STAT1, and TLR4" International Journal of Molecular Sciences 26, no. 13: 6240. https://doi.org/10.3390/ijms26136240
APA StyleCremades-Jimeno, L., López-Ramos, M., Fernández-Santamaría, R., De Pedro, M. Á., Mahillo, I., Rosales-Ariza, C., Olaguibel, J. M., Pozo, V. d., Caballero, M. L., Luna-Porta, J. A., Quirce, S., Barroso, B., Betancor, D., Valverde-Monge, M., Sastre, J., Baos, S., & Cárdaba, B. (2025). Molecular Study from the Signaling Pathways of Four Potential asthma triggers: AKT1, MAPK13, STAT1, and TLR4. International Journal of Molecular Sciences, 26(13), 6240. https://doi.org/10.3390/ijms26136240