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Gerald Thiel Gerald Thiel Intracellular signalling cascades associated with TRP channels Current state of investigations and potential applications Saarland University, Germany Campus Saarbrücken Department of Medical Biochemistry and Molecular Biology TRP channels are non-selective cation channels Ahern, 2013 The family of TRP channels IUPHR/BPS Guide to pharmacology Transient Receptor Potential (TRP) channels share a similar modular structure The cytoplasmic termini of TRP channels contain different structural and function domains that vary between subfamilies. Metabotropic and sensory TRP channels Zhang et al., 2018 TRPV1 agonists Hyperforin activates TRPC6 channels Hypericum perforatum Johanniskraut St. Johns wort Millepertuis perforé stimulus Ca2+ Na+ Na+ Hierba de San Juan Na+ , Ca2+ Ca2+ 2+ TRPC6 Ca Ca2+ cell 1 2 3 4 5 6 membrane A A Ca2+ A Na+ Ca2+ A Ca2+ Ca2+ Research in the TRP channel field - the use of different approaches TRP channels are polymodal signaling molecules Zhang et al., 2018 Stimulus-induced transcriptional activation Secondary response genes (SRGs) immediate early genes (IEGs) changing the phenotype of the cells: signalling, cell cycle, apoptosis transcription factors delayed early genes (DEGs) 100 supression of IEGs MKPs c-Fos NAB1/2 50 c-Jun Egr-1 Expression level of (% max.) level Expression 0 0 1 2 3 4 5 Time (hours) - Growth factors - Stress factors - TRP channel activators Stimulation of TRPC6 channels activates the transcription factor AP-1 Coll.luc Dimer of two basic region leucine zipper LTR HIV-1 WPRE LTR transcription factors (e.g. c-Jun/c-Fos) DU3 Flap DU3 TRE TATA luciferase wt TGAGTCA mut TGATAGT collagenase -517 +63 promotor Coll.luc Coll.luc∆TRE 10 *** 8 6 1 activity - 4 AP fold induction) fold 2 (relative activity, (relative luciferase 0 + + Hyp [1 µM] Stimulation of TRPM3 channels activates the transcription factor AP-1 Coll. Coll luc DTRE.luc 10 *** 8 6 1 activity - 4 (fold induction)(fold AP 2 rel. luciferase activity luciferaserel. 0 + + PregS [20mM] Pregnenolone sulfate +Tet CREB - a major player in stimulus- transcription coupling Hyperforin activates CREB- regulated transcription via TRPC6 c-FosCRE4.luc LTR HIV-1 WPRE LTR DU3 Flap DU3 4x CRE TATA luciferase TGACGTTT T6.11 cells c-FosCRE4.luc 6 *** 4 fold induction) fold 2 regulated transcription - relative activity, relative luciferase ( CRE 0 + Hyp [1 µM] Pregnenolone sulfate stimulates the biosynthesis of the transcription factor Egr-1 via TRPM3 activation Signal transduction- from the plasma membrane to the nucleus Stimulation of TRPV1 channels with TRPV1-ligands increases the intracellular Ca2+ concentration TRPC6 and TRPV1-induced activation of AP-1 requires extracellular Ca2+ T6.11 cells Coll.luc 10 8 6 1 actvity 1 - 4 AP (fold activation) (fold 2 relative luciferase activity luciferase relative 0 - + - + Hyperf. [1µM] - Ca2+ Essential role of Ca2+ in TRPM3-mediated upregulation of Egr-1 expression in insulinoma cells TRPM3-mediated expression of Egr-1 in insulinoma cells requires L-type voltage-gated Ca2+ channels Cross-talk of TRPM3 with voltage-gated Ca2+ channels in insulinoma cells Signal transduction- from the plasma membrane to the nucleus Essential role of Raf in TRPM3-mediated expression of Egr-1 in insulinoma cells The kinase extracellular signal- regulated protein kinase (ERK1/2) functions as a signal transducer P-ERK1/2 ERK1/2 0 0.2 0.5 1 3 5 [h] Cap. [10 µM] P-ERK1/2 ERK1/2 0 10 30 60 180 [min] PregS [20 µM] + Tet Signal transduction- from the plasma membrane to the nucleus Essential role of ERK1/2 in TRPM3- mediated upregulation of Egr-1 expression in insulinoma cells MKP-1 impairs TRPM3-induced activation of Egr-1 and AP-1 Coll.luc mock MKP-1 40 *** 30 20 1 activity - (fold induction)(fold AP 10 rel. luciferase activity luciferaserel. 0 + + PregS [20mM] +Tet Identification of secondary response genes Secondary response genes (SRGs) immediate early genes (IEGs) changing the phenotype of the cells: signalling, cell cycle, apoptosis transcription factors delayed early genes (DEGs) 100 supression of IEGs MKPs c-Fos NAB1/2 50 c-Jun Egr-1 Expression level of (% max.) level Expression 0 0 1 2 3 4 5 Time (hours) - Growth factors - Stress factors - TRP channel activators Calcitonin gene-related peptide 37-amino acid neuropeptide is a potent vasodilator showing proinflammatory activity. A main source of CGRP are TRPV1- expressing primary sensory neurons associated with pain processes. Lehninger,2000 TRPV1 stimulation induces calcitonin gene- related peptide expression via CREB Capsaicin induces the release of CGRP from sensory neurons. TRP channels are nociceptors in primary afferent sensory neurons A nociceptor is capable of sensing noxious mechanical, thermal, or chemical stimuli that give rise to pain sensation, in the periphery and transmitting the pain signal to the CNS. Sensitization of nociceptors by tissue injury and inflammation Nociceptor activation on primary sensory neurons leads to neurotransmitter release from central terminals in the spinal cord and from stimulated peripheral terminals. Some sensory neurons release the neuropeptides substance P and CGRP, which activate nearby peripheral cells. As a result, a mixture of proinflammatory factors are released, leading to nerve fiber-initiated (neurogenic) inflammation. TRP channels as analgesic targets TRPV1-deficient mice showed reduced thermal hyperalgesia after inflammation and injury. TRPV1 antagonists triggered pain relief in various models of nerve injury. First generation of TRPV1 antagonists caused hyperthermia, reflecting a role of TRPV1 in the regulation of the core body temperature Second generation of TRPV1 antagonists eliminate hyperthermia, while preserving analgesic activity. TRPM3 channels play a role in heat perception and pain responses. TRPM3 inhibitors reduced the sensitivity of mice to chemical pain and noxious heat, without altering the body temperature. Noxious heat sensing is mediated by TRPV1, TRPA1, and TRPM3 Targeting a single TRP channel might provide an insufficient effect. Vandewauw et al., 2018 Molecular neurosurgery via application or injection of capsaicin or RTX Use of TRPV1 agonists to alleviate chronic painful conditions (diabetic neuropathy, postherpetic and HIV-induce neuropathic pain). Ablation of primary sensing neurons by capsaicin or RTX Capsaicin and RTX induce calcium cytotoxicity and deletes TRPV1-expressing cells. Interleukin-8 IL-8 is a chemokine of 72 amino acids induces chemotaxis of leukocytes at sites of acute inflammation promoter of angiogenesis and tumor progression IL-8 plays a major role in the initiation and maintenance of inflammatory responses. TRP channels in pancreatic -cells TRPM3-induced Egr-1 binds to the Pdx-1 gene and increases insulin expression in insulinoma cells Activation of TRPM3 channels stimulates insulin release In TRPM3-deficient mice, basal glucose homeostasis is not critically affected. Vriens et al., 2011 Held et al., 2015 TRPM5 acts as a positive regulator of glucose-induced insulin release TRPM5 is a sodium-selective ion channel in the plasma membrane. TRPM5 is directly activated by a rise in intracellular Ca2+. Is TRPM5 a unique target for the treatment of type II diabetes ? Gram et al., 2017 Are TRP channels targets for an anticancer therapy ? TRPM3 promotes tumor growth in renal carcinoma cells. Inhibition of TRPC6 channels reduces proliferation and invasion of non-small lung cancer cells. TRPM7 channels are overexpressed in pancreatic cancer cells. TRPM8 channels are overexpressed in prostate cancer cells. TRPM8 expression appears to correlate with the grade of prostate cancer. The TRPM8 channel in prostate cancer cells is insensitive to icilin Coll.luc LNCaP 16 n.s. RT-PCR + RT - RT 12 1 activity 1 M8 M8 8 - - - AP (fold activation) (fold 4 HEK293 HEK293 LNCaP LNCaP HepG2 HepG2 LNCaP LNCaP HepG2 HepG2 HEK293 HEK293 HEK293 HEK293 relative luciferase activity activity luciferase relative 0 TRPM8 - + Icilin [10 nM] Coll.luc HEK293 HEK293-M8 16 *** 12 1 activity 1 - 8 AP (fold activation) (fold 4 relative luciferase activity activity luciferase relative 0 - + - + Icilin [10 nM] Summary TRP channels regulate many physiological functions with a strong impact on signal transduction and sensing. Plant-derived or synthetic compound are selective activators of particular TRP channels. Stimulation of TRP channels leads to the activation of stimulus- responsive transcription factors Signal transduction is mediated by a rise in intracellular Ca2+ and the activation of the protein kinases Raf and ERK. The identification of delayed response genes may explain the functions of particular TRP channels “A need for smarter drugs“ to target TRP channels for therapy. Acknowledgement Thanks to TRPM3 Sabine Meyer Isabelle Müller Andrea Lesch Sandra Rubil TRPV1 Maxi Backes TRPM8 Myriam Ulrich Jennifer Welck Anna-Lena Fischer Oliver G. Rössler.
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