Yang Xu¹*, Carole Parent^1,2,3, Pierre Coulombe^2,3,4

¹ Department of Pharmacology, University of Michigan Medical School

² Department of Cell and Developmental Biology, University of Michigan Medical School

³ Rogel Cancer Center, University of Michigan Medical School

⁴ Department of Dermatology, University of Michigan Medical School

Inflammation is a key driver and modulator of the response of skin to various types of aggression. In particular, neutrophils are the first immune cells to reach inflamed sites and have been reported to contribute to the pathogenesis of many inflammatory skin diseases such as psoriasis, skin cancers, etc. Yet, little is known about the dynamics of neutrophil infiltration in inflamed skin in vivo, and the source and identity of signals responsible for modulating neutrophil recruitment. Our project aims to visualize the spatiotemporal kinetics of neutrophil infiltration in skin in vivo after acute and recurrent irritation, and investigate whether epidermal keratinocyte-derived signals impact the amplitude and pattern of neutrophil recruitment after stress. We used a mouse dermatitis model induced by topical 12-O-tetradecanoyl phorbol-13-acetate (TPA) to study the kinetics of neutrophil recruitment to inflamed skin. We found that repeated TPA irritation to mouse skin causes amplified and prolonged neutrophil infiltration. To examine the role of keratinocyte-derived signals, we used mice carrying a null allele in the gene encoding keratin 17 (K17), an intermediate filament protein expressed in stress-activated keratinocytes. We previously reported that the expression of several pro-inflammatory genes in keratinocytes is K17-dependent. Following repeated TPA irritation, we found that K17 deficiency significantly delayed and prolonged neutrophil recruitment in skin. Additionally, intraepithelial neutrophil infiltration was specifically observed in K17null epidermis, but not in wildtype epidermis. Overall, these results suggest that K17-expressing, activated keratinocytes of the epidermis are involved in mediating the spatiotemporal pattern of neutrophil recruitment under repeated irritation conditions.

Dawn S. Kuszynski^1,2*, Barbara D. Christian¹, Anne M. Dorrance³ and D. Adam Lauver¹

¹Department of Pharmacology and Toxicology, College of Veterinary Medicine,

Michigan State University, East Lansing, MI USA

²Institute of Integrative Toxicology, Michigan State University, East Lansing, MI USA

³Department of Pharmacology and Toxicology, College of Osteopathic Medicine,

Michigan State University, East Lansing, MI USA

Clopidogrel is an effective P2Y₁₂ antagonist used to prevent arterial thrombosis. However, its use is associated with adverse bleeding events. Clinical studies have demonstrated that clopidogrel users have an increased risk of intracerebral hemorrhage. Recent reports from our laboratory and others suggest that the adverse bleeding associated with clopidogrel may not be due to the inhibition of platelets alone. To elucidate the non-platelet effects of clopidogrel treatment, we used pressure myography to measure changes in vascular function. Male New Zealand white rabbits (2.0-2.7kg) were treated with vehicle or 10mg/kg clopidogrel for three days by oral gavage. On the 4^th day, the middle cerebral arteries (MCA) were isolated and mounted in a pressure myograph. Cumulative concentrations (10^-9-10^-5M) of selective purinergic receptor agonists were administered through the MCA lumen, and the artery diameter was tracked using computer software. Analysis of MCAs from vehicle-treated rabbits demonstrated constriction in response to P2Y₂, P2Y₄, P2Y₆, and P2Y₁₄ activation. Three days of clopidogrel treatment did not affect the contraction induced by P2Y₄, P2Y₆, and P2Y₁₄ activation. However, clopidogrel treatment reduced the P2Y₂ mediated contraction (Δ diameter: vehicle, -22.486% +/- 2.107 vs. 10mg/kg clopidogrel, -14.159% +/- 3.785; p < 0.01). Removal of the endothelium revealed that endothelial P2Y₂ receptors were responsible for the constriction observed upon receptor activation. These data suggest that clopidogrel inhibits P2Y₂-mediated vasoconstriction in the MCA, which might damage distal capillaries due to abnormally high blood flow. The results provide a mechanistic explanation for the adverse cerebral bleeding associated with the drug.

Carly E. Martin*^1,2, Andrew S. Murray^1,2, Kimberley E. Sala-Hamrick¹, Jacob R. Mackinder¹, Evan C. Harrison¹ and Karin List^1,2

Departments of Pharmacology¹ and Oncology², Wayne State University

Breast cancer (BCa) and colorectal cancer (CRC) remain significant public health burdens, despite advances in detection and therapeutics. The discovery of targetable biomarkers is therefore critical to mitigate the morbidity and mortality associated with these diseases. Cancer progression is often accompanied by increased expression of pericellular proteases that degrade the extracellular matrix and cleave pro-oncogenic signaling molecules. TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, has emerged as a potential biomarker and therapeutic target. Prior data indicates that TMPRSS13 is significantly upregulated in BCa and CRC at the transcript and protein levels. Furthermore, TMPRSS13-deficient mice have few phenotypic defects but in a genetic model of mammary cancer, TMPRSS13 deficiency reduces both primary tumors and metastatic lesions. However, little is known about the biochemical and pro-oncogenic properties of TMPRSS13. Our lab discovered that TMPRSS13 is post-translationally modified by asparagine (N)-linked glycosylation, autoproteolytic cleavage, and phosphorylation, all of which are interconnected and play roles in the catalytic activity, cell-surface localization, and oncogenic properties of TMPRSS13. Site-directed mutagenesis studies of N-linked glycosylation sites and putative cleavage sites on the extracellular domain of TMPRSS13 have revealed that glycosylation at N400/N440 and cleavage at R223 of TMPRSS13 precede its autoproteolytic zymogen activation. Furthermore, abrogating glycosylation or cleavage precludes intracellular phosphorylation of TMPRSS13 and disrupts its cell surface localization. The overarching goals of this project are to understand how the unique biochemical properties of TMPRSS13 contribute to its enzymatic function and to elucidate the pro-oncogenic pathways in which TMPRSS13 plays a role.

Xue Mei¹, Blair Mell¹, Saroj Chakraborty¹, Xi Cheng¹, Ji-youn Yeo¹, Rachel Golonka¹, Piu Saha¹, Yuan Tian², Andrew D. Patterson², Matam Vijay-Kumar¹, Tao Yang¹ and Bina Joe¹

¹ Microbiome Consortium, Center for Hypertension and Precision Medicine, Department of Physiology and Pharmacology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, USA

² Department of Veterinary & Biomedical Sciences, The Pennsylvania State University, University Park, PA, USA

Hypertensive subjects present with alterations in gut microbiota and short chain fatty acids (SCFAs). SCFAs are the major metabolic products of bacterial fermentation in the intestine. While acetate and propionate are energy sources for peripheral tissues, butyrate is the primary energy source for the host colonic epithelium via β-oxidation or glycolysis. Since butyrate is known to elevate blood pressure (BP), we hypothesized that gut microbial butyrate-mediated elevation in BP is linked with an increased proximal colon energy metabolism. To test this hypothesis, 7 weeks old concomitantly raised male germ-free Sprague Dawley rats (GF, n=5-6) were compared with GF acquiring microbiota for 10 days (GFC, n=6) for BP, cecal butyrate by proton nuclear magnetic resonance, microbial profiling by 16S RNA sequencing and proximal colonic transcriptomic signatures for energy metabolism by RT-qPCR. GFC rats represented an energy-repleted state with a marked increase of butyrate. Interestingly, GFC rats had a significant increase in systolic and diastolic BP compared to GF rats (p<0.05). This increase in BP was associated with a significant upregulation of 11 genes (Pparγ, Ffar2, Acss1, Acadl, Cpt2, Hk2, Pfkp, Pgam1, Gpd2, Sirt3, Crat, p≤0.05) for energy metabolism pathways (β-oxidation and glycolysis) in the colon of the GFC rats compared to GF rats. Overall, our work presents the first evidence for a direct relationship between host energy metabolism at the proximal gut-microbiota interface and BP regulation. Further, butyrate, the predominant colonic energy metabolite, may contribute to the mechanism governing this relationship between energy metabolism in the proximal colon and BP regulation.

Tao Yang¹, Ethel Tackie-Yarboi², Xue Mei¹, Jun Kyoung¹, Blair Mell¹, Ji-Youn Yeo¹, Xi Cheng1, Isaac T. Schiefer², Bina Joe¹

¹Microbiome Consortium, Center for Hypertension and Precision Medicine, Department of Physiology and Pharmacology

²Shimadzu Laboratory for Pharmaceutical Research Excellence, Department of Medicinal and Biological Chemistry, University of Toledo

Our previous studies demonstrated that AEC inhibitor interacts with gut microbiota to regulate blood pressure. Gut microbiota harbors esterase, which may result in ACE inhibitor quinapril hydrolysis in the gut prior to absorption. The current study is to test the hypothesis that gut microbiota modulates the efficacy of quinapril.

16 weeks old male Spontaneously Hypertensive Rats (SHR) were orally gavaged with (N=12) or without (N=6) Vancomycin, Meropenem, and Omeprazole (VMO) at 50 mg/kg/day for five days to deplete the gut microbiota. To test the efficacy of Quinapril, a single dose of 8mg/kg quinapril was orally administered and BP was recorded via radio-telemetry. Quinapril catabolism was quantitated by Liquid chromatography–mass spectrometry. Bacterial esterase activity was monitored by the hydrolysis of p-nitro-phenylbutyrate. Cecal microbiota was analyzed by 16S rRNA.

With 50% reduction in bacterial 16S copy number (P<0.0001), the SHR+VMO group had (1) reduced Eggerthellaceae (P<0.0001), an esterase-harboring bacterial family; (2) a lower cecal esterase activity to hydrolyze quinapril (P=0.0065); (3) a lower catabolic activity for quinapril, determined by the reduction in quinapril quantity after incubation with cecal lysate (P<0.0001); (4) decreased genes in drug metabolism in KEGG pathways (P<0.0001). Importantly, administration of quinapril to the SHR+VMO resulted in a significant lowering of BP, compared to SHR (P=0.0015).

Our study is the first to reveal a previously unrecognized, novel role of gut microbiota in antihypertensive drug resistance in vivo, which expands the prospect for clinical management of resistant hypertension by manipulating gut microbiota.

Nicholas Denomme¹*, Samantha L. Hodges¹, Alan V. Smrcka¹ and Lori L. Isom¹

Department of Pharmacology,

University of Michigan, Ann Arbor, MI, USA.

G protein βγ subunits can directly modulate the activity of voltage-gated K⁺ and Ca²⁺ ion channels, however, less well-known is their ability to modulate voltage-gated sodium channel (VGSC) function. VGSCs are transmembrane protein complexes responsible for the initiation and propagation of action potentials in neurons. VGSC subtypes are strategically expressed based on their kinetic and voltage-sensing properties to optimize physiological function.

The Na_v1.6 subtype is the most abundant VGSC expressed in the adult brain. The sensitivity of Na_v1.6 to modulation by Gβγ subunits is unknown. We hypothesized based on the sequence similarity of Na_v1.6 to known Gβγ targets Na_v1.1 and Na_v1.2 that it would show significant functional modulation by Gβγ subunits. Indeed, transient coexpression of Gβ1γ2 in HEK cells stably expressing human Na_v1.6 led to a significant decrease in peak whole cell sodium current density. Additionally, coexpression of Gβ1γ2 in HEK cells stably expressing human Na_v1.1 also led to a significant decrease in current density. In contrast to Nav1.6, the inhibition of Na_v1.1 displayed voltage-dependence, with a significant current decrease occurring from a prepulse potential of -70 mV, but not -120 mV. No shifts in the voltage-dependence of activation or inactivation of Na_v1.1 or Na_v1.6 were induced by the coexpression of Gβ1γ2 subunits.

Our work shows that the major neuronal VGSC subtypes Na_v1.6 and Na_v1.1 are relevant effectors of Gβ1γ2 signaling. Given the vital role of Gβγ subunits in GPCR-mediated neurotransmission and Na_v1.6 in controlling neuronal excitability, these data motivate further characterization of this functional interaction in native neuronal preparations.