Previously, immunostaining of lung tissue from 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, we tested responses of human small airway epithelial cells to the SARS-CoV-2 spike protein receptor binding domain (SPRBD). We found increased expression of carbohydrate sulfotransferases (CHST) 11 and 15 which lead to increases in chondroitin 4-sulfate and chondroitin sulfate E and reduced expression of ARSB. We investigated the underlying transcriptional mechanisms and identified increases in phospho-p38 MAPK and phospho-SMAD3 following exposure to the SPRBD. Inhibition of p38 MAPK or SMAD3 suppressed the promoter activation of CHST15 and CHST11. Decline in ARSB expression was mediated by a novel mechanism in which phospho-p38α induced N-terminal Rb phosphorylation. This was associated with increased Rb-E2F1 binding and decline in E2F1 binding to the ARSB promoter. This link between Rb and ARSB expression may help to explain how ARSB is involved in fundamental regulation of cell proliferation. 

In addition to effects of phospho-p38-MAPK inhibitors and the SMAD3 inhibitor S IS3, treatment with the antihistamine desloratadine and the antibiotic monensin impacted on the CHST and ARSB expression in the lung cells. Also, in lung tissue from the mouse model of carrageenan-induced systemic inflammation, increases in phospho-p38 MAPK and expression of CHST15 and CHST11 and declines in ARSB expression and ARSB promoter DNA-E2F binding occurred. These effects are 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 the effects of Covid-19 and other infections.

Several clinical trials have considered the role of antihistamines in Covid-19 infection. The current study findings of an effect on  pathways which can lead to increased production of chondroitin sulfates and decline in ARSB indicate a mechanism through p38alpha which may explain benefit of antihistamine.  Clearly, additional studies are required to define the precise steps by which the SPRBD-ACE2 interaction can activate p38alpha and how histamine receptors are involved.

The novel findings on Rb-E2F1 interaction provide critical new insight into how ARSB expression may be regulated. Many inhibitors of ARSB are known (high salt, low CFTR, ethanol, carrageenan, androgen, hypoxia), so it is of profound interest to identify that changes in Rb phosphorylation and activation may regulate ARSB expression. We have identified decline in ARSB in malignant cells and tissues; these declines may follow Rb inactivation. The underlying mechanism mediated by p38 was presented by Gubern et al (PMID: 27642049), with clear presentation of effects on N-terminal Rb phosphorylation.

Other investigators have shown that an impaired response of patients with moderate COPD to oxygen was associated with deficient ARSB expression (PMID: 30353303). This result is consistent with the impact of hypoxia on ARSB which we reported (PMID:22428001).  Decline in ARSB mimicked effects of hypoxia in bronchial and intestinal epithelial cells (PMID:22428001), leading to decline in HIF-1alpha and other changes in gene expression. Hypoxia, like ARSB silencing, significantly increased the chondroitin 4-sulfate and total sulfated glycosaminoglycan content. Correlation between Ct values of hypoxia and ARSB silenced was r=0.994. We have suggested that the relationship between ARSB and oxygen may predispose to aerobic glycolysis (Warburg effect), due to impaired utilization of mitochondrial sulfate when ARSB is deficient (PMC:5015117).

This relationship between effects of hypoxia and decline in ARSB may help to understand the pathobiology in the Covid-19 lung and other conditions in which there is pulmonary fibrosis. In our previous publication (PMIS:34788765), immunohistochemistry showed decline in intensity and distribution of ARSB in Covid-19 lungs, as well as increased chondroitin sulfate and increased perivascular CHST15. Changes were also evident in lung tissue with diffuse alveolar damage from other causes.

Perhaps most interesting and relevant to pathobiology associated with decline in ARSB, either from SARS-CoV-2 infection or other exposure, is the impact on chondroitin 4-sulfation. The only established function of ARSB is to remove the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate or dermatan sulfate. However, this change in sulfation was shown to affect the binding of galectin-3 (less binding when ARSB is reduced and C4S sulfation is increased; PMID:24240681) and SHP2 (more binding when ARSB is reduced and C4S is increased; PMID:27078017, PMID:29794138). These changes in galectin-3 and SHP2 activation have significant downstream effects, modulating proliferation, signaling, and differentiation through both genetic and epigenetic effects.

We hope that there will be increased attention to the role of sulfatases, chondroitin sulfation, sulfotransferases, and sulfated glycosaminoglycans. This attention may lead to new insights and new treatments with meaningful impact on health and disease.