SARS-CoV-2 spike protein-ACE2 interaction increases carbohydrate sulfotransferases and reduces N-acetylgalactosamine-4-sulfatase by p38 MAPK

Abstract

Immunostaining in lungs of patients who died with COVID-19 infection showed increased intensity and distribution of chondroitin sulfate and decline in N-acetylgalactostamine-4-sulfatase (Arylsulfatase B; ARSB). To explain these findings, human small airway epithelial cells were exposed to the SARS-CoV-2 spike protein receptor binding domain (SPRBD) and transcriptional mechanisms were investigated. Phospho-p38 MAPK and phospho-SMAD3 increased following exposure to the SPRBD, and their inhibition suppressed the promoter activation of the carbohydrate sulfotransferases CHST15 and CHST11, which contributed to chondroitin sulfate biosynthesis. Decline in ARSB was mediated by phospho-38 MAPK-induced N-terminal Rb phosphorylation and an associated increase in Rb-E2F1 binding and decline in E2F1 binding to the ARSB promoter. The increases in chondroitin sulfotransferases were inhibited when treated with phospho-p38-MAPK inhibitors, SMAD3 (SIS3) inhibitors, as well as antihistamine desloratadine and antibiotic monensin. In the mouse model of carrageenan-induced systemic inflammation, increases in phospho-p38 MAPK and expression of CHST15 and CHST11 and declines in DNA-E2F binding and ARSB expression occurred in the lung, similar to the observed effects in this SPRBD model of COVID-19 infection. Since accumulation of chondroitin sulfates is associated with fibrotic lung conditions and diffuse alveolar damage, increased attention to p38-MAPK inhibition may be beneficial in ameliorating Covid-19 infections.

Fig. 1

Chondroitin sulfate, total sulfated glycosaminoglycans, Arylsulfatase B, sulfotransferase activity, and expression of chondroitin sulfotransferases and other enzymes for chondroitin biosynthesis. a Corresponding to the findings in the infected lung tissue, total chondroitin sulfate (CS) increased by ~2 ug/g protein (p = 8.1 × 10⁻⁵, n = 6) and total sulfated glycosaminoglycans increased by over 3 ug/g protein (sGAG) (p = 0.0005, n = 6) in the AEC following exposure to the SPRBD. Increases are more following combined treatment with SPRBD and IFN-β (>5 µg/g protein for CS, n = 6; 7.7 µg/g protein for sGAG, n = 6). b Consistent with the observed increases in chondroitin sulfate in Covid-19 lung tissue and in the airway cells, sulfotransferase activity increased by 63% following SPRBD and by over 200% following the combination of SPRBD and IFN-β (n = 6, n = 6). c Arylsulfatase B (ARSB) activity declined in the AEC following exposure to the SPRBD (p = 0.0006, n = 6), and declined over 50% by the combined exposure to SPRBD and IFN-β (p = 7.1 × 10⁻⁸, n = 6). d The mRNA expression of ARSB also declined (p = 6.6 × 10⁻⁵, n = 6) following SPRBD, and declined further following exposure to the combination of SPRBD and IFN-β. e Expression of both CHST15 and of CHST11 is significantly upregulated following exposure to the SPRBD and the combination of SPRBD and IFN-β. f Expression of the enzymes PAPSS1, PAPSS2, and CHSY1, which are required for chondroitin biosynthesis, increased significantly following exposure to SPRBD (p ≤ 2.4 × 10⁻⁶, n = 6) with further increase following IFN-β and SPRBD (p ≤ 3.4 × 10⁻⁶, n = 6). All p-values were determined using unpaired t tests, two-tailed, with unequal variance, and error bars represent one standard deviation. *** represents p ≤ 0.001, and **** is for p ≤ 0.0001. ARSB arylsulfatase B = N-acetylgalactosamine-4-sulfatase, CHST carbohydrate sulfotransferase, CHSY1 chondroitin sulfate synthase, CS chondroitin sulfate, IFN interferon, PAPSS 3’-phosphoadenosine 5’-phosphosulfate synthase, sGAG sulfated glycosaminoglycan, SPRBD spike protein receptor-binding domain

Fig. 2

SMAD3 and p38 MAPK inhibitors abrogate the effects of SPRBD. a SIS3, a specific inhibitor of phospho-SMAD3, inhibited the SPRBD- and SPRBD + IFN-β- induced increases in expression of CHST15 and CHST11. b Following silencing of ACE2 by siRNA, the SPRBD- and SPRBD + IFN-β- induced increases in phospho-(Thr180/Tyr182)-p38 MAPK were inhibited. c The SPRBD- and SPRBD + IFN-β- induced increases in phospho-(S423/S425)-SMAD3 were completely inhibited by SIS3 and, to a lesser extent, by SB, the p38 MAPK inhibitor. d The SPRBD and SPRBD + IFN-β- induced increases in phospho-(Thr180/Tyr182)-p38 were unaffected by exposure to SIS3 and were inhibited by SB. e, f Promoter activity of CHST15 and CHST11 was enhanced by exposure to SPRBD and to a greater extent by the combination of IFN-β and SPRBD. Both SIS3 and SB inhibited promoter activation. g Treatment of cells with the specific phospho-p38α/β inhibitor PH797804 or with p38α siRNA completely blocked the SPRBD-induced increase in phospho-p38 MAPK detected by ELISA. In contrast, TAB1 siRNA and control siRNA had no effect. h The SPRBD-induced increases in expression of CHST15 and of CHST11 were inhibited by p38α siRNA and by PH797804, but not by TAB1 siRNA or control siRNA. All p-values were determined by unpaired t test, two-tailed, with unequal variance, and with at least three independent experiments. Error bars represent one standard deviation. *** represents p ≤ 0.001; **** is for p ≤ 0.0001. ACE angiotensin-converting enzyme, CHST carbohydrate sulfotransferase, NSC NSC23766, SB SB203580, si siRNA, SIS3 specific inhibitor of SMAD3, SMAD Mothers against decapentaplegic homolog 3 (DPC3), SPRBD spike protein receptor-binding domain, TAB1 TGF-beta activated kinase (MAP3K7)

Fig. 3

Spike protein receptor binding domain inhibits ARSB activity and expression by activation of phospho-p38 MAPK and phospho-(S249/T252)-RB-E2F1 interaction. a ARSB mRNA expression was unaffected by NSC23766, but following exposure to the p38 MAPK inhibitor SB, mRNA expression was restored to baseline control values. b In contrast, to the observed effects of p38α inhibitors on expression of CHST11 and CHST15, p38α siRNA and PH797804 reversed the SPRBD-induced decline in ARSB expression. TAB1 siRNA had no effect. c SIS3 had no impact on the SPRBD- or SPRBD + IFN-β- induced decline in ARSB expression. d ARSB promoter activation was reduced by SPRBD and by SPRBD + IFN-β. These declines were reversed by exposure to SB, but not by SIS3. e Following treatment with SPRBD or SPRBD + IFN-β, C-terminal phospho-(Ser807/811)-Rb declined as shown by ELISA. These declines were reversed by SB, but not by SIS3. f In contrast to the decline in C-terminal Rb phosphorylation, exposure to SPRBD + IFN-β increased N-terminus phospho-(S249/T252)-Rb, as detected by Western blot and shown in detail in Supplementary Fig. 4a–d. This increase was inhibited by SB, and total Rb was unchanged. g Densitometry confirms the impression of Western blot and shows the ratio of phospho-(S249)-Rb to total Rb following SPRBD + IFN-β has increased to 3.89 times the baseline. h Following exposure to SPRBD and SPRBD + IFN-β, E2F-DNA binding declined significantly, and SB reversed the declines. i %DNA input declined following SPRBD + IFN-β and increased following inhibition of p38-MAPK by SB. j Agarose gel of chromatin immunoprecipitation (ChIP) indicated no effect of the IgG negative control on E2F1 binding to the ARSB promoter at baseline, increased binding following IFN-β, reduced binding following SPRBD + IFN-β, and reversal of this decline following treatment with SB. P values were determined by unpaired t tests, two-tailed with unequal variance, with n of at least 3 independent experiments. Error bars show one standard deviation. * represents p ≤ 0.05; ** is for p ≤ 0.01, *** for p ≤ 0.001, and **** for p ≤ 0.0001. ARSB arylsulfatase B = N-acetylgalactosamine-4-sulfatase, IFN interferon, ND no difference, NSC = NSC23766, Rb retinoblastoma protein, SB = SB203580, SIS3 specific inhibitor of Smad3, SPRBD spike protein receptor-binding domain

Fig. 4

Effects of desloratadine, monensin, and carrageenan on expression of CHST15, CHST11, and ARSB. a Exposure to the antihistamine desloratadine at a concentration of 32 µM for 2 h reduced by 62% the SPRBD-induced increase in phospho-(Thr180/Tyr182)-p38 MAPK in the AEC. b Consistent with the decline in phospho-p38 MAPK, desloratadine reduced the SPRBD- and SPRBD + IFN-β- induced increases in CHST15 (p = 0.04; p ≤ 0.0003 with IFN-β) and CHST11 expression (p = 0.0008; p ≤ 0.0009 with IFN-β, n = 6). Declines increased with increasing concentrations of desloratadine from 16 µM to 32 µM to 64 µM, without evidence of cytotoxicity. c Fold-change of ARSB mRNA expression increased significantly and progressively with desloratadine 16–64 µM for 2 h (p = 0.02, p = 1.35 × 10⁻⁶, p = 4.3 × 10⁻⁸ with IFN-β; n = 6). d Desloratadine 32 µM for 2 h partially reversed the SPRBD- and SPRBD + IFN-β- induced declines in ARSB activity (from 64.7 to 73.4 nmol/mg protein/h and from 41.0 to 61.8 nmol/mg protein/h with IFN-β; n = 3) (p = 0.036, p = 0.0042, n = 3). e The polyether antibiotic monensin 1.0 µM for 2 h reduced the SPRBD-induced increases in CHST15 mRNA (p = 0.005; p ≤ 0.0001 with IFN-β) and CHST11 (p = 0.002, p ≤ 0.0002 with IFN-β; fold-change compared to control; n = 6). f Monensin had no significant impact on the SPRBD-induced decline in ARSB. g Carrageenan-exposed C57BL/6 J mice had marked increase in phospho-p38 MAPK in lung tissue (p = 6.0 × 10⁻⁶, n = 6 per group). h mRNA expression of CHST15 and CHST11 was significantly increased in the lung tissue of the carrageenan-exposed mice (p < 1 × 10⁻⁵, n = 6). In contrast, ARSB expression declined significantly (p = 8.4 × 10⁻⁹, n = 6). i Consistent with the phospho-38 MAPK-Rb-E2F1-mediated mechanism of regulation of ARSB mRNA expression, the DNA-bound E2F was significantly reduced (p = 1.2 × 10⁻⁶, n = 6) in the lung tissue of the carrageenan-exposed mice. All p values were determined using unpaired t tests, two-tailed, with unequal variance, and error bars represent one standard deviation. * represents p ≤ 0.05; ** is for p ≤ 0.01, *** for p ≤ 0.001, and **** for p ≤ 0.0001. ARSB arylsulfatase B = N-acetylgalactosamine-4-sulfatase, CHST carbohydrate sulfotransferase, Des Desloratadine, IFN interferon, Mon Monensin, ND no difference, PAPSS 3’-phosphoadenosine 5’-phosphosulfate synthase, sGAG sulfated glycosaminoglycan, SPRBD spike protein receptor-binding domain

Fig. 5

SARS-CoV-2 spike protein binding with ACE2 initiates transcriptional events which modify chondroitin sulfation through activation of phospho-p38 MAPK. This schematic presents the signaling mechanism by which the SARS-CoV-2 spike protein receptor-binding domain (SPRBD) interacts and initiates the transcriptional events which lead to increased chondroitin sulfate in airway epithelial cells and in Covid-19 infected lung. The activation of phospho-p38 MAPK and phospho-SMAD3 lead to increased expression of CHST15 and CHST11. Phospho-p38 MAPK leads to N-terminal phosphorylation of Rb and enhanced Rb binding with E2F1, which reduces ARSB promoter binding with E2F1 and reduces ARSB expression. Additional effects may arise from ACE2-SPRBD binding, due to reduced availability of ACE2 for other biochemical reactions, including the conversion of AngII to Ang1-7. AngII interaction with the angiotensin II receptor type 1 (AT1R) increases phospho-p38 MAPK, and increased availability of AngII may contribute to the observed downstream effects of p38 MAPK on transcription. The altered balance between AngII-induced vasoconstriction and Ang1-7 vasodilatation, following interaction with the G-protein coupled Mas receptor, may have additional pathophysiological consequences. ACE2 angiotensin-converting enzyme 2, AngII angiotensin II, ARSB arylsulfatase B, AT1R angiotensin II receptor type 1, AT2R angiotensin II receptor type 2, CHST carbohydrate sulfotransferase, Mas Ang1-7 receptor, pRb retinoblastoma protein, SMAD Suppressor of Mothers against Decapentaplegic, SPRBD SARS-CoV-2 spike protein receptor binding domain