Design and Evaluation of Heterobivalent PAR1–PAR2 Ligands as Antagonists of Calcium Mobilization

Majewski, Mark W.; Gandhi, Disha M.; Rosas, Ricardo; Dockendorff, Chris

doi:10.26434/chemrxiv.6394430.v1

Cited by 1 publication

(1 citation statement)

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“…PARs are known to participate in diverse processes, including thrombosis, pain, and inflammation (Mrozkova et al, 2016;Crilly et al, 2012;McCulloch et al, 2018;Heuberger et al 2019). Notably, PAR2 is being investigated as a therapeutic target for pain and inflammation and many modulators of PAR2 have been developed so far (Ramachandran et al 2012;Ferrell et al, 2008;Yau et al, 2013;Yau et al 2016;Jiang et al, 2018;Lee et al, 2019;Majewski et al, 2018).…”

Section: Supplementarymentioning

confidence: 99%

Sustained exposure to trypsin causes cells to transition into a state of reversible stemness that is amenable to transdifferentiation

Sharma

Kumar

Sharma

et al. 2019

Preprint

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During cell culture, trypsin, a serine protease, is applied to cells for 5-10 minutes to separate them from each other and from the underlying substratum so that they can be transferred to a different vessel, for re-plating, after growth medium containing 10 % serum has been added to the cells, in a well-known technique known as 'passaging'. The serum in the growth medium contains alpha-1 antitrypsin, which is a potent inhibitor of trypsin, elastase and other serine proteases.Although what is used is bovine serum in which levels of proteins could be different from levels seen in humans, normal human serum contains A1AT (> 1 mg/ml; > ~18 µmol/L) as well as trypsin itself (< 460 ng/ml, or ~0.02 µmol/L), with the former in a ~900-fold molar excess over the latter. Thus, it may be assumed there is also enough A1AT in the bovine serum added during passaging, to neutralize the trypsin (~100 μM) present in the small volume of trypsin-EDTA solution used to separate cells. What are the consequences of not adding serum, when growth medium is added, or of maintaining cells for a few tens of hours in the presence of trypsin, in a serum-free growth medium? What does such sustained exposure to trypsin during cell culture do to cells? More generally, what are the responses of cells within an organism to the balance of trypsin and A1AT in the serum that bathes them constantly? We know that excesses and deficiencies in the levels of either trypsin or A1AT are associated with disease. We know that cellular metabolism can be influenced through signaling involving protease activated membrane GPCR receptors (PAR1-4). In particular, we know of a receptor called PAR2, which is specifically activated by trypsin, expressed by cells at baseline levels, and upregulated through some feedback involving trypsin-activation. We also know that cells at sites of injury or inflammation produce and secrete trypsin, and that this trypsin can act locally upon cells in a variety of ways, all of which have probably not yet been elucidated. Here, we show that sustained exposure to trypsin induces cells to de-differentiate into a stem-like state. We show that if serum is either not added at all, or added and then washed away (after confluency is attained), during cell culture, all cells exposed to exogenously-added trypsin undergo changes in morphology, transcriptome, secretome, and developmental potential, and transition into a state of stemness, in minimal essential medium (MEM). Regardless of their origins, i.e., independent of whether they are derived from primary cultures, cell lines or cancer cell lines, and regardless of the original cell type used, exposure to trypsin (~10 µM; ~250 µg/ml) at a concentration 10-fold lower than that used to separate cells during passaging (~100 μM), over a period of 24-48 hours, causes cells to (1) become rounded, (2) cluster together, (3) get arrested in the G0/G1 stage of the cell cycle, (4) display increased presence of 5-hydroxymethyl cytosine in their nuclei (indicative of reprogramming), (5) display increased...

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