Toll-like receptor 4 (TLR4) and protease-activated receptor 2 (PAR2) play pivotal roles in the mammalian innate immune response. Notably, in addition to their involvement in detection of invading ...pathogens, PAR2 and TLR4 modulate the levels of cell death-induced sterile inflammation by activating pro- or anti-inflammatory downstream signaling cascades. Within the central nervous system, there is emerging evidence that both receptors are involved in synaptic transmission and brain plasticity. Furthermore, due to their prominent role in mediating neuroinflammation, PAR2 and TLR4 are associated with development and progression of neurodegenerative disorders including but not limited to Alzheimer's disease, Parkinson's disease and multiple sclerosis. In this article, we summarise the current knowledge on the cooperation between PAR2 and TLR4, discuss the potential cross-talk levels and highlight the impact of the cross-coupling on neuroinflammation.
Exacerbated sensitivity to mechanical stimuli that are normally innocuous or mildly painful (mechanical allodynia and hyperalgesia)
occurs during inflammation and underlies painful diseases. ...Proteases that are generated during inflammation and disease cleave
protease-activated receptor 2 (PAR 2 ) on afferent nerves to cause mechanical hyperalgesia in the skin and intestine by unknown mechanisms. We hypothesized that
PAR 2 -mediated mechanical hyperalgesia requires sensitization of the ion channel transient receptor potential vanilloid 4 (TRPV4).
Immunoreactive TRPV4 was coexpressed by rat dorsal root ganglia (DRG) neurons with PAR 2 , substance P (SP) and calcitonin gene-related peptide (CGRP), mediators of pain transmission. In PAR 2 -expressing cell lines that either naturally expressed TRPV4 (bronchial epithelial cells) or that were transfected to express
TRPV4 (HEK cells), pretreatment with a PAR 2 agonist enhanced Ca 2+ and current responses to the TRPV4 agonists phorbol ester 4α-phorbol 12,13-didecanoate (4αPDD) and hypotonic solutions. PAR 2 -agonist similarly sensitized TRPV4 Ca 2+ signals and currents in DRG neurons. Antagonists of phospholipase Cβ and protein kinases A, C and D inhibited PAR 2 -induced sensitization of TRPV4 Ca 2+ signals and currents. 4αPDD and hypotonic solutions stimulated SP and CGRP release from dorsal horn of rat spinal cord, and
pretreatment with PAR 2 agonist sensitized TRPV4-dependent peptide release. Intraplantar injection of PAR 2 agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4αPDD and hypotonic solutions.
Deletion of TRPV4 prevented PAR 2 agonist-induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR 2 activates a second messenger to sensitize TRPV4-dependent release of nociceptive peptides and induce mechanical hyperalgesia,
may underlie inflammatory hyperalgesia in diseases where proteases are activated and released.
The COVID-19 pandemic created an unprecedented global healthcare emergency prompting the exploration of new therapeutic avenues, including drug repurposing. A large number of ongoing studies revealed ...pervasive issues in clinical research, such as the lack of accessible and organised data. Moreover, current shortcomings in clinical studies highlighted the need for a multi-faceted approach to tackle this health crisis. Thus, we set out to explore and develop new strategies for drug repositioning by employing computational pharmacology, data mining, systems biology, and computational chemistry to advance shared efforts in identifying key targets, affected networks, and potential pharmaceutical intervention options. Our study revealed that formulating pharmacological strategies should rely on both therapeutic targets and their networks. We showed how data mining can reveal regulatory patterns, capture novel targets, alert about side-effects, and help identify new therapeutic avenues. We also highlighted the importance of the miRNA regulatory layer and how this information could be used to monitor disease progression or devise treatment strategies. Importantly, our work bridged the interactome with the chemical compound space to better understand the complex landscape of COVID-19 drugs. Machine and deep learning allowed us to showcase limitations in current chemical libraries for COVID-19 suggesting that both in silico and experimental analyses should be combined to retrieve therapeutically valuable compounds. Based on the gathered data, we strongly advocate for taking this opportunity to establish robust practices for treating today's and future infectious diseases by preparing solid analytical frameworks.
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•A new computational pharmacology perspective on compound development and drug repurposing highlighting the need to incorporate diverse compound screening libraries with a strong understanding of their interactome and regulome. Introducing the concept of ‘the chemical space as a proxy to model biological interactions’.•Analysis of the current trends and shortcomings in COVID-19 drug repurposing demonstrating the value of the computational pharmacology perspective.•The introduced in-depth analysis revealed the importance of expanding clinical studies beyond direct drug-target interactions in order to consider a more complex space of the affected networks.•The findings of miRNAs networks offer a new strategy to search for valuable biomarkers or therapeutic management options.•The study demonstrated that the chemical space for COVID-19 investigational compounds might not be broad enough and could benefit from additional experimental evidence to create more robust models.
G protein-coupled receptors (GPCRs) are important cell signaling mediators, involved in essential physiological processes. GPCRs respond to a wide variety of ligands from light to large ...macromolecules, including hormones and small peptides. Unfortunately, mutations and dysregulation of GPCRs that induce a loss of function or alter expression can lead to disorders that are sometimes lethal. Therefore, the expression, trafficking, signaling and desensitization of GPCRs must be tightly regulated by different cellular systems to prevent disease. Although there is substantial knowledge regarding the mechanisms that regulate the desensitization and down-regulation of GPCRs, less is known about the mechanisms that regulate the trafficking and cell-surface expression of newly synthesized GPCRs. More recently, there is accumulating evidence that suggests certain GPCRs are able to interact with specific proteins that can completely change their fate and function. These interactions add on another level of regulation and flexibility between different tissue/cell-types. Here, we review some of the main interacting proteins of GPCRs. A greater understanding of the mechanisms regulating their interactions may lead to the discovery of new drug targets for therapy.
Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR1 and PAR4, ...the endogenous enzymes responsible for activating PAR2 in settings other than the gastrointestinal system, where trypsin can activate PAR2, are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR1, PAR2, and PAR4; 2) PAR-dependent calcium signaling responses in cells expressing PAR1, PAR2, and PAR4 and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR4-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR1, PAR2, and PAR4 and to disarm/inhibit PAR1. Although hK5 and -6 were also able to activate PAR2, they failed to cause PAR4-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR2-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR1, PAR2, and PAR4.
The putative cache (Ca
2+
channel and chemotaxis receptor) domain containing 1 (CACHD1) protein has predicted structural similarities to members of the α2δ voltage-gated Ca
2+
channel auxiliary ...subunit family. CACHD1 mRNA and protein were highly expressed in the male mammalian CNS, in particular in the thalamus, hippocampus, and cerebellum, with a broadly similar tissue distribution to Ca
V
3 subunits, in particular Ca
V
3.1. In expression studies, CACHD1 increased cell-surface localization of Ca
V
3.1, and these proteins were in close proximity at the cell surface, consistent with the formation of CACHD1-Ca
V
3.1 complexes. In functional electrophysiological studies, coexpression of human CACHD1 with Ca
V
3.1, Ca
V
3.2, and Ca
V
3.3 caused a significant increase in peak current density and corresponding increases in maximal conductance. By contrast, α2δ-1 had no effect on peak current density or maximal conductance in Ca
V
3.1, Ca
V
3.2, or Ca
V
3.3. A comparison of CACHD1-mediated increases in Ca
V
3.1 current density and gating currents revealed an increase in channel open probability. In hippocampal neurons from male and female embryonic day 19 rats, CACHD1 overexpression increased Ca
V
3-mediated action potential firing frequency and neuronal excitability. These data suggest that CACHD1 is structurally an α2δ-like protein that functionally modulates Ca
V
3 voltage-gated calcium channel activity.
SIGNIFICANCE STATEMENT
This is the first study to characterize the Ca
2+
channel and chemotaxis receptor domain containing 1 (CACHD1) protein. CACHD1 is widely expressed in the CNS, in particular in the thalamus, hippocampus, and cerebellum. CACHD1 distribution is similar to that of low voltage-activated (Ca
V
3, T-type) calcium channels, in particular to Ca
V
3.1, a protein that regulates neuronal excitability and is a potential therapeutic target in conditions such as epilepsy and pain. CACHD1 is structurally an α2δ-like protein that functionally increases Ca
V
3 calcium current. CACHD1 increases the presence of Ca
V
3.1 at the cell surface, forms complexes with Ca
V
3.1 at the cell surface, and causes an increase in channel open probability. In hippocampal neurons, CACHD1 causes increases in neuronal firing. Thus, CACHD1 represents a novel protein that modulates Ca
V
3 activity.
There is strong evidence that the omega-3 polyunsaturated fatty acids (n-3 PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have cardioprotective effects. n-3 PUFAs cause ...vasodilation in hypertensive patients, in part controlled by increased membrane conductance to potassium. As K
channels play a major role in vascular tone regulation and are involved in hypertension, we aimed to verify whether n-3 PUFA-mediated vasodilation involved the opening of K
channels. We used a murine model in which the K
channel pore subunit, Kir6.1, is deleted in vascular smooth muscle. The vasomotor response of preconstricted arteries to physiologically relevant concentrations of DHA and EPA was measured using wire myography, using the channel blocker PNU-37883A. The effect of n-3 PUFAs on potassium currents in wild-type native smooth muscle cells was investigated using whole-cell patch clamping. DHA and EPA induced vasodilation in mouse aorta and mesenteric arteries; relaxations in the aorta were sensitive to K
blockade with PNU-37883A. Endothelium removal didn't affect relaxation to EPA and caused a small but significant inhibition of relaxation to DHA. In the knock-out model, relaxations to DHA and EPA were unaffected by channel knockdown but were still inhibited by PNU-37883A, indicating that the action of PNU-37883A on relaxation may not reflect inhibition of K
. In native aortic smooth muscle cells DHA failed to activate K
currents. We conclude that DHA and EPA cause vasodilation in mouse aorta and mesenteric arteries. Relaxations in blocker-treated arteries from knock-out mice demonstrate that K
channels are not involved in the n-3 PUFA-induced relaxation.
Inflammatory proteases (mast cell tryptase and trypsins) cleave protease-activated receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown ...mechanisms. We determined whether transient receptor potential vanilloid receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2 was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2 agonists increased intracellular Ca2+ (Ca2+i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2 and TRPV1. PAR2 agonists potentiated capsaicin-induced increases in Ca2+i in TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in Ca2+i and TRPV1 currents. Activation of PAR2 or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2 agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2 agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2 activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2 sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia.
Due to the ageing population, there is a steadily increasing incidence of osteoporosis and osteoporotic fractures. As conventional pharmacological therapy options for osteoporosis are often ...associated with severe side effects, bone grafts are still considered the clinical gold standard. However, the availability of viable, autologous bone grafts is limited making alternative cell-based strategies a promising therapeutic alternative. Adipose-derived stem cells (ASCs) are a readily available population of mesenchymal stem/stromal cells (MSCs) that can be isolated within minimally invasive surgery. This ease of availability and their ability to undergo osteogenic differentiation makes ASCs promising candidates for cell-based therapies for bone fractures. Recent studies have suggested that both exposure to electrical fields and cultivation in 3D can positively affect osteogenic potential of MSCs. To elucidate the osteoinductive potential of a combination of these biophysical cues on ASCs, cells were embedded within anionic nanofibrillar cellulose (aNFC) hydrogels and exposed to electrical stimulation (ES) for up to 21 days. ES was applied to ASCs in 2D and 3D at a voltage of 0.1 V/cm with a duration of 0.04 ms, and a frequency of 10 Hz for 30 min per day. Exposure of ASCs to ES in 3D resulted in high alkaline phosphatase (ALP) activity and in an increased mineralisation evidenced by Alizarin Red S staining. Moreover, ES in 3D aNFC led to an increased expression of the osteogenic markers osteopontin and osteocalcin and a rearrangement and alignment of the actin cytoskeleton. Taken together, our data suggest that a combination of ES with 3D cell culture can increase the osteogenic potential of ASCs. Thus, exposure of ASCs to these biophysical cues might improve the clinical outcomes of regenerative therapies in treatment of osteoporotic fractures.
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