Histamine signaling and metabolism identify potential biomarkers and therapies for lymphangioleiomyomatosis (2021)
Figure 5

Figure 5. Histamine-mediated signaling and metabolism in LAM.

A Overabundance of histamine in Tsc2 wild-type relative to Tsc2-deficient MEFs, both growth in DMEM 10% FBS. The asterisks indicate significant difference with two-sided t-test (P = 0.017); replicates/condition n = 5, and independent experiments n = 2). Average values are indicated with lilac-colored lines.

B Isotope profiling of [13C6,15N3]-labeled L-histidine measured by LC-MS/MS in Tsc2-deficient and wild-type MEFs grown in DMEM 10% FBS without unlabeled L-histidine. Labeled-measured metabolites are depicted in the X-axis. The significant differences correspond to two-sided t-test (*P = 0.032 and **P = 0.008; replicates/condition n = 5). Average values are indicated with lilac-colored lines.

C Fold-change variation of histamine and MIAA levels measured by LC-MS/MS assays in rapamycin-exposed (20 nM, 16 hours) Tsc2-deficient MEFs relative to DMSO-exposed for the same period. The significant difference corresponds to one-sided t-test (P = 0.012; replicates/condition n = 4 and independent experiments n = 2). Average values are indicated with lilac-colored lines.

D Western blot results of HRH1, SLC22A3, and loading control from Tsc2-deficient and wild-type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10% or 0.5% FBS (independent experiments n = 2). The expression levels are indicated by the ratio of the corresponding signal relative to loading control and basal setting (noted as 1(ref)).

E Representative images of immunohistochemical detection of HRH1 in LAM lung lesions. A total of seven LAM patients were analyzed and assay controls are shown in Appendix Fig. S2.

F Evaluation of cell viability inhibition by loratadine alone (concentrations shown on X-axis, from 0 to 100 μM) or combined with everolimus (fixed to 20 nM) in Tsc2-deficient and wild-type MEF cultures grown in DMEM 10% FBS for 72 hours. The synergistic combination index (CIx < 1) is shown (replicates/condition n = 3, independent experiments n = 3). Each data point represents the mean and SD.

G Percentages of viability of Tsc2-dificient MEFs exposed to increasing concentrations of histamine (X-axis) in DMEM 0.5% FBS without L-histidine for 72 hours. The asterisk indicates significant difference with one-sided t-test (P = 0.040; replicates/condition n = 4-6, independent experiments n = 2). The bars indicate mean ± SD.

H Percentages of viability of Tsc2-dificient and wild-type MEFs exposed to DMEM 10% or 0.5% FBS with or without L-histidine for 72 hours. The asterisks indicate significant differences with one-sided t-test (10% FBS, P = 0.017; 0.5% FBS, P = 5x10-4; replicates/condition n = 4-6, independent experiments n = 2). The bars indicate mean ± SD.

I Western blot results of phospho-Tyr783 PLCγ1 and loading control in Tsc2-deficient MEFs grown in DMEM 0.5% FBS without L-histidine and exposed to histamine (1 μM) for 10 minutes or 1 hour. The signals of phospho-Tyr783 PLCγ1 relative to loading control or non-phospho PLCγ1, and to basal setting (noted as 1(ref)) are indicated.