SARS-CoV-2 spike protein-mediated cell signaling in lung vascular cells

Currently, the world is suffering from the pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses angiotensin-converting enzyme 2 (ACE2) as a receptor to enter the host cells. So far, 30 million people have been infected with SARS-CoV-2, and nearly 1 million people have died because of COVID-19 worldwide, causing serious health, economical, and sociological problems. However, the mechanism of the effect of SARS-CoV-2 on human host cells has not been defined. The present study reports that the SARS-CoV-2 spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in lung vascular cells. The treatment of human pulmonary artery smooth muscle cells or human pulmonary artery endothelial cells with recombinant SARS-CoV-2 spike protein S1 subunit (Val16 – Gln690) at 10 ng/ml (0.13 nM) caused an activation of MEK phosphorylation. The activation kinetics was transient with a peak at 10 min. The recombinant protein that contains only the ACE2 receptor-binding domain of SARS-CoV-2 spike protein S1 subunit (Arg319 – Phe541), on the other hand, did not cause this activation. Consistent with the activation of cell growth signaling in lung vascular cells by SARS-CoV-2 spike protein, pulmonary vascular walls were found to be thickened in COVID-19 patients. Thus, SARS-CoV-2 spike protein-mediated cell growth signaling may participate in adverse cardiovascular/pulmonary outcomes, and this mechanism may provide new therapeutic targets to combat COVID-19.

Thus, managing the pulmonary and cardiovascular aspects of COVID-19 is considered to be the key for reducing the severity of COVID-19 and the associated mortality. However, it is unclear exactly how SARS-CoV-2 affects humans. Thus, understanding the mechanism of SARS-CoV-2 actions should help develop therapeutic strategies to reduce the mortality and morbidity associated with COVID-19.
The SARS-CoV-2 spike protein is critical to initiate the interactions between the virus and the host cell surface receptor and facilitates the viral entry into the host cell by assisting in the fusion of the viral and host cell membranes [11,12]. This protein consists of two subunits: Subunit 1 (S1) that contains the ACE2 receptor-binding domain (RBD) and Subunit 2 (S2) that is responsible for fusion. 4 The current dogma of the SARS-CoV-2 mechanism is that the binding of viral spike protein to the host ACE2 receptor results in the entry of virus into the host cells and the cellular response is a result of the viral infection [5,6,11,12]. However, we herein show that SARS-CoV-2 spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in human host cells, suggesting a novel biological mechanism of SARS-CoV-2 actions.

Cell culture
Human pulmonary artery smooth muscle cells and human pulmonary artery endothelial cells were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and rat pulmonary artery smooth muscle cells were purchased from Cell Applications (San Diego, CA, USA). Cells were cultured in accordance with the manufacturers' instructions in 5% CO 2 at 37°C. Cells at passages 3-6 were maintained in low fetal bovine serum (0.4%)-containing medium overnight before the treatment as routinely performed in experiments on cell signaling and protein phosphorylation [13].
Cells were treated with the recombinant SARS-CoV-2 spike protein full length S1 subunit that contains most of the S1 subunit (Val16 -Gln690) with a molecular weight of 75 kDa (RayBiotech, Peachtree Corners, GA, USA) or the recombinant SARS-CoV-2 spike protein RBD (RayBiotech) that only contains the RBD region (Arg319 -Phe541) with a molecular weight of 25 kDa. Some cells were pretreated for 1 h with the rabbit anti-ACE2 antibody (Catalog # 4355; Cell Signaling Technology, Danvers, MA). 5

Statistical Analysis
For Western blotting data, means and standard errors of mean (SEM) were computed. Two groups were compared by a two-tailed Student's t test, and differences between more than two groups were determined by the analysis of variance (ANOVA). p < 0.05 was defined to be statistically significant. For the morphometric analysis, IBM SPSS Statistics software version 22.0 was used for the statistical calculations. Mann-Whitney U Test was used to define the statistical significance at p < 0.05.

SARS-CoV-2 spike protein alone can activate cell signaling in human cells
To test the hypothesis that SARS-CoV-2 spike protein alone can elicit cell signaling, human pulmonary artery smooth muscle cells were treated with the full length S1 subunit (Val16 -Gln690) of the SARS-CoV-2 spike protein for 0, 10, and 30 min. As shown in Fig. 1A, the spike protein at a concentration of 10 ng/ml (0.133 nM) strongly activated the phosphorylation of 45 kDa MEK at Ser217 and Ser221 residues. The kinetics of MEK phosphorylation promoted by the full-length S1 subunit (Val16 -Gln690) of the SARS-CoV-2 spike protein was consistently found to be transient with a peak at 10 min. This fast activation suggests that this may be a receptor-mediated cell-signaling event. Similarly, full-length S1 subunit SARS-CoV-2 spike protein promoted the phosphorylation of MEK in human pulmonary artery endothelial cells (Fig.   1B). SARS-CoV-2 spike protein, however, did not activate other signaling events such as Akt (Fig. 1A) and Stat3 (Fig. 1B) pathways.
As we performed experiments to determine the effects of the full-length S1 subunit of SARS-CoV-2 spike protein on rat pulmonary artery smooth muscle cells, we surprisingly found that, not only SARS-CoV-2 spike protein did not promote the MEK phosphorylation, but rather decreased the phosphorylation. As shown in Fig. 2, the treatment of full length SARS-CoV-2 spike protein S1 resulted in the dephosphorylation of MEK as early as 10 min after the treatment and this dephosphorylation event was maintained for at least 60 min. Thus, SARS-CoV-2 spike protein promotes the MEK phosphorylation in human cells, but not in rat cells.
To confirm that the action of SARS-CoV-2 spike protein is through its well-known receptor ACE2, 5,6 human pulmonary artery smooth muscle cells were pretreated with the ACE2 antibody for 1 hour before treating with the full-length S1 subunit of SARS-CoV-2 spike protein. 8 The ACE2 antibody alone caused the activation of MEK, and SARS-CoV-2 spike protein did not further increase this MEK phosphorylation signal (Fig. 3).

RBD domain alone is not sufficient to activate cell signaling
To test whether the RBD binding to ACE2 is sufficient to stimulate cell signaling for MEK phosphorylation, human cells were treated with the recombinant S1 RBD of SARS-CoV-2 spike protein that only contains the RBD (Arg319 -Phe541). In contrast to the full-length S1 subunit (Val16 -Gln690) that strongly phosphorylated MEK, S1 RBD (Arg319 -Phe541) did not activate the MEK phosphorylation in human pulmonary artery smooth muscle cells (Fig. 4A) or in human pulmonary artery endothelial cells (Fig. 4B). Thus, other regions of the spike protein in addition to RBD may be required for eliciting cell signaling for MEK phosphorylation.

Pulmonary vascular walls are thickened in COVID-19 patients
Our results showing that SARS-CoV-2 spike protein is capable of activating the MEK/ERK pathway in pulmonary artery smooth muscle and endothelial cells suggest that cell growth signaling may be triggered in the pulmonary vascular walls in response to SARS-CoV-2.
To test this, we examined the lung histology results of patients who died of COVID-19.  (Fig. 5B, right).

Discussion
The major finding of this study is that the SARS-CoV-2 spike protein without the rest of the virus can elicit cell signaling, specifically the activation of the MEK/ERK pathway, in human host lung vascular smooth muscle and endothelial cells. MEK is a mitogen-activated protein kinase kinase (MAPKK) that phosphorylates and activates extracellular-regulated kinase (ERK), one type of mitogen-activated protein kinases (MAPK). In this MEK/ERK pathway of cell signaling, MEK is activated by the phosphorylation by Raf1 kinase [14]. The MEK/ERK 1 0 pathway is a well-known cell growth mechanism [15] and has also been shown to facilitate the viral replication cycles [16].
By contrast to the full-length S1 subunit of SARS-CoV-2 spike protein (Val16 -Gln690) that strongly activated MEK, RBD only containing protein (Arg319 -Phe541) did not activate MEK in human pulmonary artery smooth muscle or in endothelial cells. These results suggest that the protein regions other than the RBD (i.e. Val16 -Arg319 and/or Phe541 -Gln690) are required for eliciting cell signaling for the MEK phosphorylation. Thus, we propose that SARS-CoV-2 spike protein-mediated cell signaling is not merely cells responding to anything binding to the membrane surface protein, but is a growth factor/hormone-like specific cell signal transduction event that is well coordinated by the RBD as well as other protein regions that facilitate cell signaling. Our ACE2 antibody experiments as described in Fig. 3 support the involvement of ACE2. However, the binding of the ACE2 antibody to ACE2 seems sufficient to cause the activation of MEK. While we interpreted that the ACE2 antibody blocked and interfered with the binding of the RBD to ACE2, it is possible that ACE2 antibody-mediated cell signaling may have desensitized the subsequent spike protein-mediated effects to activate MEK.
Thus, SARS-CoV-2 spike protein-mediated cell signaling may occur through a receptor other than ACE2.
In contrast to the effects of SARS-CoV-2 spike protein on human cells to activate the MEK phosphorylation, this protein promoted the dephosphorylation of MEK in rat cells. This species difference in the SARS-CoV-2 spike protein actions may be important to understand how this virus severely affects humans. We previously reported a novel ligand-mediated dephosphorylation mechanism of MEK induced by neurotensin and neuromedin N [17]. The SARS-CoV-2 spike protein signaling in rat pulmonary artery smooth muscle cells represents 1 1 another example of the MEK dephosphorylation mechanism. These results also suggest that the species-specific actions of SARS-CoV-2 may depend on how spike protein-mediated cell signaling mechanisms differ among species.
It has been noted that elderly patients with systemic hypertension and other cardiovascular diseases are particularly susceptible to developing severe and possibly fatal conditions of COVID-19 [4,9,10]. Thus, the pathology of COVID-19 does not seem to be patients. These results also indicate the possibility that patients who recovered from COVID-19 may be predisposed to developing pulmonary arterial hypertension and right-sided heart failure.
We also noticed that published lung histology images of patients who died of ARDS during the 2002 -2004 SARS outbreak due to the infection with SARS-CoV-1 did not exhibit the signs of thickened pulmonary vascular walls [18,19]. Thus, pulmonary vascular wall thickening is a unique feature of the SARS-CoV-2 infection and COVID-19.

Conclusions
Our results in human pulmonary artery smooth muscle and endothelial cells revealed that the SARS-CoV-2 spike protein S1 subunit is sufficient to trigger biological responses in the human host cells in the absence of the participation of the rest of the SARS-CoV-2 viral particle.
We also found that the pulmonary vascular walls are thickened in COVID-19 patients. We propose that SARS-CoV-2 spike protein-mediated cell growth signaling participates in adverse cardiovascular/pulmonary outcomes seen in COVID-19. This mechanism may provide new therapeutic targets to combat the SARS-CoV-2 infection and COVID-19.

Conflicts of Interest
None.