Here, we isolated and identified by mass spectrometry the dipeptide γ-Glu-Leu as a component of a Phytophthora infestans mycelium extract that induces [Ca2+]cyt elevation. Treatment of Arabidopsis seedlings with synthetic γ-Glu-Leu revealed stimulatory effects on defense signaling, including a weak enhancement of the expression of some MAMP-inducible genes or affecting the refractory period to a second MAMP elicitation. However, γ-Glu-Leu is not a classical MAMP since pH adjustment abolished these activities and importantly, the observed effects of γ-Glu-Leu could be recapitulated by mimicking extracellular acidification. Thus, although γ-Glu-Leu can act as a direct agonist of calcium sensing receptors in animal systems, the Ca2+-mobilizing activity in plants reported here is due to acidification. Low pH also shapes the Ca2+ signature of well-studied MAMPs (e.g. flg22) or excitatory amino acids such as glutamate. Overall, this work serves as a cautionary reminder that in defense signaling studies where Ca2+ flux measurements are concerned, it is important to monitor and consider the effects of pH.Cytosolic Ca The DAMP AtPep1 is recognized by PEP RECEPTOR1/2 (PEPR1/2)10. Sensing of MAMPs/DAMPs by the PRRs activates a multicomponent signaling network. Signal transduction includes early physiological changes such as an increase in cytosolic Ca2+ and the production of reactive oxygen species (ROS), and employs phosphorylation pathways involving mitogen-activated protein kinase (MAPKs) cascades and calcium-dependent protein kinases (CPKs). These signaling processes lead to a reprogramming of gene expression, changes in phytohormone levels and other cellular signaling pathways, and ultimately in increased synthesis as well as secretion of antimicrobial substances13. The sum of all these induced defense responses constitutes the plant immune response termed pattern-triggered immunity (PTI)15.Plants possess complex defense systems to prevent invasion by potentially harmful microorganisms. Essential for the initiation of defense is the detection of the microbes through binding of so-called microbe-associated molecular patterns (MAMPs) or molecules derived from damaged plant tissue (DAMP) to specific pattern recognition receptors (PRRs). Examples for pairs of MAMP/PRR identified in Arabidopsis are flagellin (epitope flg22)/FLAGELLIN SENSITIVE2 (FLS2)2+ concentration ([Ca2+]cyt) is one of the earliest observable response to environmental alterations, where kinetic variations in the Ca2+ transients or oscillations are thought to encode information16. Such “Ca2+ signatures” are decoded by a number of plant decoding mechanisms; these include Ca2+ binding proteins such as calmodulins (CaM), calcineurin B-like (CBL) proteins and CPKs (reviewed in17). The PAMP-induced alterations in [Ca2+]cyt are dependent on the corresponding PRRs18. Reciprocally, when [Ca2+]cyt is used as a readout in a genetic screen for mutants in early PAMP responses, the predominant mutants are the PRRs themselves, components of the receptor complex20 or elements of membrane protein (i.e. receptor) ER/Golgi quality control pathways22. This finding suggests that [Ca2+]cyt measurements may be exploited to screen for PRRs of orphan MAMPs/DAMPs and as proof-of-principle for this idea, the ATP receptor23 and a putative lipopolysaccharide (LPS) receptor24 have been isolated by such a strategy.A change in cytosolic Ca2+ influx, MAMP treatment also induces movement of other ions such as K+ efflux and H+ influx across the plasma membrane25. The resulting reduction of extracellular H+ concentration leads to an apoplastic alkalinization response27. The corresponding cytosolic acidification is believed to have signaling functions intracellularly29. Similarly, a link between Ca2+ signaling and pH dynamics has been observed in studies on developmental processes like the growth of pollen tubes30 and roots34, as well as in treatment of seedlings with different stimuli like exogenous auxin35 or mechanical stimulation36. In Arabidopsis roots, for example, oscillating changes in [Ca2+]cyt, extracellular pH and ROS might act together to control growth of root hair tips as well as root gravitropism34. Recently, parallel pH and Ca2+fluorescent reporter-based imaging after application of ATP, 1-naphthaleneacetic acid or wounding to Arabidopsis seedlings demonstrated that [Ca2+]cyt and pH dynamics act in concert37. Thus, intracellular calcium and pH signaling are tightly intertwined.In addition to CaPhytophthora species are classified as Peronosporomycetes (Oomycetes), which are phylogenetically distinct from true fungi. In fact, they belong to the Stramenopiles that comprise diatoms and brown algae38. Several Phytophthora species cause devastating diseases on major crops, such as potato late blight (P. infestans), soybean root rot (P. sojae) and ramorum blight on trees (P. ramorum). P. infestans, the causative agent of potato late blight, colonizes only members of the Solanaceae family; it is not adapted to infect non-solanaceous plants. Interaction of the nonhost Arabidopsis with this pathogen is characterized by the formation of cell wall depositions at sites of attempted infection in the epidermal cell layer, and in case of successful invasion, by a hypersensitive response of the affected cells40. Initial recognition of P. infestans, or generally Phytophthora, by plants presumably involves conserved MAMP molecules but their identity has been elusive. While some Phytophthora defense-triggering MAMPs (reviewed in42) are known, reports on Phytophthora MAMPs that elicit immunity in Arabidopsis are rare. The Cellulose-Binding Elicitor Lectin (CBEL) from P. parasitica var. nicotianae is a non-enzymatic cell wall glycoprotein with two cellulose-binding domains and lectin-like activities44. CBEL not only activates defense responses in the host tobacco44, but also in the nonhost Arabidopsis45, where CBEL perception might occur indirectly via alterations of the cellulose status in the plant cell wall46. Necrosis- and ethylene-inducing peptide1-like proteins (NLPs) are microbial effectors secreted by a wide range of bacteria, fungi and Peronosporomycetes48 and it was initially unclear if plant defense is receptor-mediated or through cellular damage from tissue necrosis. Subsequently, a conserved 20-mer amino acid sequence (nlp20) present in both cytotoxic and non-cytotoxic NLPs proved to be sufficient to trigger defense responses in a variety of Brassicaceae49. In this case, recognition of nlp20 occurs through the PRR complex consisting of the receptor-like protein RLP23, the receptor-like kinase SOBIR1 (SUPPRESSOR OF BAK1-INTERACTING RECEPTOR KINASE1 (BIR1)) and BAK151.P. infestans and initiate a genetic screen based on [Ca2+]cyt measurements to identify the corresponding PRR(s). Here, we isolated and identified the dipeptide γ-Glu-Leu as a component of a P. infestans mycelium extract that induces [Ca2+]cyt elevation. Treatment of Arabidopsis seedlings with γ-Glu-Leu showed that it could promote some defense responses to MAMPs. However, pH adjustment of the dipeptide solution abolished most of the effects, highlighting the impact of acidification on defense-related signaling, a largely overlooked and underestimated phenomenon.The initial aim of our study was to isolate MAMPs from P. infestans MAMPs that trigger immune responses in its nonhost Arabidopsis, we isolated elicitor(s) extracted from liquid culture-grown mycelium following a protocol modified from Monjil et al.52. The authors reported that their extract caused growth retardation in Arabidopsis seedlings and induced an oxidative burst, hypersensitive response and defense gene expression when infiltrated into leaves, but so far there is no information available on the active component(s). When we challenged aequorin-expressing Arabidopsis (pMAQ2) seedlings with the crude P. infestans mycelium extract, we observed a transient increase in [Ca2+]cyt with a peak height of 186 ± 6 nM followed by a prolonged decline to basal level, which lasted more than 15 minutes   do not bind strongly to the octadecyl carbon chains of the reverse-phase columns and is/are likely hydrophilic in nature. To identify putative elicitor(s), this water-elutable mycelium extract fraction was subjected to an untargeted mass spectrometry (MS) analysis. Samples were measured in positive (ESI(+)) and negative (ESI(−)) ionization mode and evaluated separately. Five compounds were extracted from the ESI(+) profiles and four compounds from the ESI(−) profiles , because it exhibits characteristic MS fragments at m/z 244.11 , 215.14 , 198.11 , 170.12 , 132.1 , 130.05  resulting from neutral losses and alpha cleavages  substances and to induce a rise in intracellular [Ca2+] in taste receptor cells in the lingual tissues of mice53. These γ-glutamyl kokumi peptides appear to directly engage calcium-sensing receptor (CaSR) in the taste buds54. Thus, among the identified components in the mycelium extract, γ-Glu-Leu is a candidate Ca2+ flux-inducing compound and acts similarly in plants as an agonist of calcium channels. Alternatively, it may act as a MAMP to induce Ca2+ elevations. To test if the dipeptide γ-Glu-Leu is indeed the active component from the mycelium extract that induces the [Ca2+]cyt increase, we applied synthetic γ-Glu-Leu (ranging from 50 to 1000 µM) to aequorin-expressing Arabidopsis seedlings. Treatment with 50 µM γ-Glu-Leu had no obvious effect on the basal level of [Ca2+]cyt represented by the water control, whereas all higher concentrations induced a transient elevation in Ca2+ flux with dose-dependent magnitudes between 129 ± 7 nM [Ca2+]cyt for 100 µM and 340 ± 5 nM [Ca2+]cyt for 1000 µM γ-Glu-Leu . To test this, pMAQ2 seedlings were treated with a saturating concentration of γ-Glu-Leu (500 µM) for ~15 min prior to a second stimulation with ME. In comparison to the control (with water as pretreatment), γ-Glu-Leu severely reduced Ca2+ peaks obtained with a subsequent application of ME   Fig. . SimilarµM) Fig. . This isµM) Fig. , which iµM) Fig. . However57. We therefore tested whether a combination of γ-Glu-Leu and flg22 (as a representative MAMP) will affect Arabidopsis defense responses differently compared to single treatments. We compared the effect of single or combined stimuli on Ca2+ flux as well as relative transcript levels of several flg22-responsive defense genes. Simultaneous treatment with non-saturating concentrations of γ-Glu-Leu (250 µM) and flg22 (10 nM) induced a similar Ca2+ signature to that obtained with γ-Glu-Leu alone but with a significantly slightly higher amplitude of 255 ± 8 nM [Ca2+]cyt , CINNAMYL ALCOHOL DEHYDROGENASE5 (CAD5), FLG22-INDUCED RECEPTOR-LIKE KINASE1 (FRK1), MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3), MITOGEN-ACTIVATED PROTEIN KINASE11 (MPK11), NDR1/HIN1-LIKE10 (NHL10), PHYTOALEXIN DEFICIENT3 (PAD3), PHYTOCHELATIN SYNTHASE1 (PCS1), PHOSPHATE-INDUCED1 (PHI1), WRKY DNA-BINDING PROTEIN33 (WRKY33), WRKY DNA-BINDING PROTEIN53 (WRKY53) and ZINC FINGER PROTEIN12 (ZAT12)59. When applied alone, γ-Glu-Leu did not induce the expression of any of these genes even at the high concentration of 500 µM  or MAPK phosphorylation , it no longer induced a rise in [Ca2+]cyt and the additive effects of flg22 co-treatment on Ca2+ flux . Adding this dilute acetic acid solution to Arabidopsis seedlings indeed induced elevations in  increase in animal tissues53. Amplitude of [Ca2+]cyt increase and the kinetics of the recovery phase were dose-dependent, which is in accordance with various other stresses such as application of hydrogen peroxide62, flg2264 or AtPep163. γ-Glu-Leu also had additive effects on induction of selected flg22-responsive genes. However, γ-Glu-Leu did not (strongly) induce MAPK phosphorylation or ROS accumulation. These observations are reminiscent of recent studies involving cellulose-derived cellobiose fragments acting as a DAMP. Cellobiose weakly activated MPK6 but does not trigger production of ROS or callose deposition; however, it synergistically enhances Arabidopsis defenses in co-treatment with flg2265. We also tested if pre-treatment with γ-Glu-Leu would induce resistance to subsequent bacterial infection  but this was not the case (not shown). Thus, γ-Glu-Leu is not a MAMP.Our original goal was to isolate novel 2+]cyt recovery to resting level was decreased and an apparent second rise in [Ca2+]cyt, resembling the beginning of some form of Ca2+ oscillation, appeared . We could abrogate the biological effects of γ-Glu-Leu by buffering the pH exogenously and more importantly, mimicked these effects by applying weak acids like acetic acid Fig. . We had red Figs  and S3..WRKY33 retained the boosting effect of γ-Glu-Leu  or salicylic acid, which are all substances that may lower external pH if not sufficiently buffered. Also clustered to the low pH dataset were transcriptomes of flg22 treatment (1 hpi) and P. infestans infection (6 and 12 hpi). For the latter, one may speculate that the pH effect of γ-Glu-Leu present on the mycelium may have shaped the P. infestans infection transcriptome. For flg22, the correlation between low pH and flg22 response is actually in agreement with our observation that the normal Ca2+ response to flg22 is dampened when flg22 is MES-buffered 74, which have been implicated in Ca2+ fluxes induced by flg22, elf18 and chitin75. While Glu is well established as a second messenger that can mobilize Ca2+ through iGluRs, it should be noted that most studies in plants required millimolar concentrations of Glu to elicit [Ca2+]cyt changes77. The recent work on Glu as a systemic wound signal used 100 mM Glu to induce [Ca2+]cyt changes and determined apoplastic Glu to reach 50 mM at the damage sites. However, such high concentrations of Glu will certainly involve cellular acidification. Indeed, comparing MES-buffered  to unbuffered Glu revealed that Glu-induced Ca2+ fluxes may be largely overestimated if pH is not taken into consideration . Taken together, our current study validates a known but often overlooked fact that apoplastic acidification can induce a rise in [Ca2+]cyt79.As mentioned above, we initially speculated that the acidic Glu moiety within γ-Glu-Leu would be responsible for the acidification-induced Caing Fig. , suggesttin Fig. . Compareent Fig. . The reaP. infestans mycelium extract and can induce Ca2+ flux in Arabidopsis seedlings. Although it can act as a direct agonist of calcium sensing receptor in animal systems, the mechanistic of its Ca2+-mobilizing action is unknown in plants. More importantly, all of its elicitor-like properties in plants appear to be due to acidification (presumably apoplastic), and, thus, suggest that γ-Glu-Leu is not a MAMP. Nevertheless, its presence during infection may still contribute to signaling processes that are sensitive to pH alterations. Notably, pH also contributes to the Ca2+ signature of established peptide MAMPs such as flg22 or excitatory amino acids such as glutamate. Overall, our work serves as a cautionary reminder that in studies of defense response and  where plant Ca2+ flux measurements are concerned, control of pH is crucial but this is, unfortunately, not always true in the literature.γ-Glu-Leu is found in 80. Seeds were surface-sterilized in 12-well plates (~20 seeds per well) and stratified at 4 °C for 3–5 days in 2 ml liquid MS medium per well . Seedlings grew under long day conditions  at 20–22 °C for 7–10 days.The Arabidopsis line pMAQ2 was used in all experiments. pMAQ2 expresses cytosolic p35S-apoaequorin in a Col-0 backgroundP. infestans isolate CRA208m281 was cultivated in 20 ml (per 100 ml Erlenmeyer flask) of liquid oat bean medium  in the dark at 18 °C. After 19–22 days, mycelium mats were harvested and thoroughly washed with water. Remaining water was filtered through a nylon mesh placed in a Buchner funnel. For extraction, the protocol of Monjil et al.52 was modified. Mycelium was ground in liquid nitrogen with a Retsch mill  and subsequently homogenized in methanol (1 ml methanol/1 g mycelium) for 2 min using a Polytron . After centrifugation  1 ml aliquots of the supernatant were evaporated in a SpeedVac (Thermo Fisher Scientific). Pellets were resolved in 1 ml water and combined. To remove salts, extract was loaded onto a Chromabond C18 silica column , which was treated with methanol prior to use. The flowthrough fraction was discarded and extract components eluted with water . Thus, 1 ml of extract derived from 100 mg of mycelium. Mycelium and extract were stored at −20 °C.8 were synthesized on a ResPep SL peptide synthesizer . Shrimp shells (Sigma-Aldrich) were used for chitin treatment. Stock solutions of the peptides (1 mM) and shrimp shell chitin (100 mg shrimp shell/ml) were prepared with water, dilutions of the stocks with water, 5 mM MES buffer (pH 6.0) or acetic acid .Flg22, elf18 and AtPep1Julius-Kühn-Institut, Berlin). γ-Glu-Ile was synthesized as described in the following. For peptide coupling, protected glutamic acid , HOBt  and EDC  were suspended in dry CH2Cl2 (10 ml) and stirred at 0 °C for 15 min. Isoleucine t-butyl ester hydrochloride  was added, then DIPEA  was syringed in one portion and the resulting solution was stirred at room temperature overnight (~12 h). The reaction mixture was diluted with 100 ml EtOAc, transferred to a separation funnel and sequentially washed with 0.5 M aqueous citric acid (2 × 50 mL), saturated aqueous NaHCO3 (2 × 50 ml) and brine (1 × 30 ml). The organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. To remove the Boc/OtBu protection groups, the crude peptide was exposed to high vacuum for 1 h before dissolving it in a mixture 3:1 DCM/TFA (5 ml). Pressure from gas evolution generated during the dissolving process was regularly relieved by opening the reaction flask. After 3 h, no starting material was detected by thin layer chromatography and ESI-MS. The volatiles were fully removed under reduced pressure and the resulting thick oily residue was dried at high vacuum for 2 h. Boc-γ-Glu(OtBu)-Ile-OtBu was recovered at a yield of 81% .The dipeptides γ-Glu-Leu and α-Leu-Glu were purchased from Bachem. Leu-Phe, Leu-Ile, Phe-Leu, Tyr-Ile and Ile-Ile were kindly provided by Christoph Böttcher . All other dipeptides were dissolved in water (as a 100 mM stock solution), and diluted accordingly with water or 5 mM MES (pH 6.0).82. Ca2+ concentrations were calculated as described in Rentel and Knight83.Aequorin luminescence measurements were performed as whole seedling assays with individual seedlings (7–10 days old) in 96-well microplatesPROTEIN PHOSPHATASE 2A SUBUNIT A3, PP2AA3) were performed as described in Maldonado-Bonilla et al.84. The primers used are listed in Table UBC9 and UBC21), the PP2AA3 gene was tested to be non-responsive to γ-Glu-Leu or apoplastic acidification  coupled to a MicrOTOF–Q I hybrid quadrupole time-of-flight mass spectrometer. For this purpose, samples were ionized at positive ESI(+) and negative ESI(−) ionization using an Apollo II electrospray ion source . All mass spectra were acquired in centroid mode and recalibrated on the basis of lithium formate cluster ions. DataAnalysis 4.2  was used for the generation of extracted ion chromatograms, deconvolution of mass spectra and calculation of elemental compositions. γ-Glu-Leu was quantified using Quant Analysis 4.2.Mycelium extracts (20-fold concentrated) were measured on an Acquity UPLC system (Waters, 50 value of γ-Glu-Leu and statistical analyses were performed with the software GraphPad Prism 5.0. Details of the statistical analysis are specified in figure legends.Calculation of the ECSupplementary Info- Westphal et al


Here, we review recent work in this area, assess the potential of in vivo heat generation by this mechanism, and advocate that a comparative approach is the best path toward resolving many outstanding questions on the physiological function, regulation, and evolution of SLN.A regulatory protein (sarcolipin or SLN) bound to SERCA pumps in the sarcoplasmic reticulum (SR) of cardiac and skeletal muscle has been shown to increase heat production 2+ levels underlies the modulation of optimal contraction strengths, frequencies, and relaxation rates by cardiac and skeletal muscle over a wide range of activities. This regulation is largely achieved by metering the release of SR Ca2+ stores into the cytosol via ryanodine receptor channels and the subsequent uptake of Ca2+ by sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps. Briefly, SERCA harnesses the phosphate bond energy of one ATP molecule to translocate two Ca2+ ions into the SR lumen, thereby generating a ~15,000-fold (1.5 mM vs. 0.1 μM) lumen-to-cytosol Ca2+ concentration gradient in resting muscle  helical peptide that was initially shown to alter Ca2+ uptake kinetics by inhibiting SERCA1a and SERCA2a activity in the atria and skeletal muscles of mammals . While this work is tantalizing, several key mechanistic aspects remain unanswered such as (1) whether uncoupling of Ca2+ transport from ATP hydrolysis in SERCA primarily arises from slippage of the SERCA-bound Ca2+ ions into the cytosol after ATP hydrolysis or from passive leak of luminal Ca2+ back into the cytosol through SERCA .Precise control of cytosolic CaA Figure , (2) whade Meis, . Also un2+ turnover during a muscle contraction-relaxation cycle. These include activation of sarcolemma Na+-K+-ATPases for membrane repolarization, myosin-ATPases for cross-bridge relaxation, and SERCA Ca2+-ATPases for Ca2+ sequestration , with an additional 750–1,167 ATP hydrolyzed by the Na+-K+ and myosin ATPases  by up to 35% .Unfortunately, UCP1  have nonetheless evolved extreme cold tolerance while others  are notoriously cold intolerant (even often as adults). In contrast to such expectations, not only is the SLN locus functionally intact in each of these cases , but its primary structure has remained exceptionally well conserved despite highly divergent body sizes and thermoregulatory capacities among species. For example, UCP1-lacking elephants, whales, pigs, and hyraxes all possess in vitro, taken together, the above findings argue that the primary role of SLN in skeletal muscle of mammals is not thermoregulatory in nature. Nonetheless, SLN expression and in vitro studies on a range of large-bodied mammals, including species lacking functional UCP1, are required to confirm or refute the above contentions. An examination of representative marsupials and monotremes are also required to assess the thermogenic potential of SLN across Mammalia and better pinpoint if and when the proposed thermogenic function arose in evolutionary time. Finally, it is worth noting that the high expression level of SLN in mammalian atria—the concentration of which is 10-to 1,000-fold higher than in the soleus of rodents  Ca2+ affinity of SERCA. Indeed, both elevated and lowered atrial SLN concentrations are linked to cardiac pathologies  studies are also required to elucidate what component of the SLN peptide is involved in thermogenesis. Two recent studies have implicated the cytosol-oriented N-terminus to underlie Ca2+ uncoupling , although only Glu7 was predicted as necessary to induce the uncoupling structural change . By contrast, the luminal C-terminal sequence (27RSYQY31)—which underlies both the proper SR/ER cellular localization of SLN and its apparent inhibition of SERCA  that are proposed to be essential for SLN inhibition , while this C-terminal residue is deleted in equids  within the cytosolic domain have been shown to relieve the inhibitory effect of SLN on cardiac SERCA pumps —universally present from fish to mammals  also lack the potential for reversible phosphorylation  or non-mammalian vertebrates  is also without empirical evidence and remains purely speculative. Comparative studies taking advantage of naturally occurring variability in SLN concentrations and primary sequence among vertebrates provide ideal avenues to explore the function of this regulatory peptide, and its evolution.While there can be little doubt that the interaction of SLN with SERCA lowers the metabolic efficiency of CaKC drafted the manuscript, prepared the figures, and approved the final version. AD contributed to manuscript writing, provided important interpretations, critically revised the work, and provided final approval of the opinion content.AD was employed by Fish & Richardson P.C. at the time of writing. The ideas and opinions in the article are her own, and predominantly arose from biophysical research on SLN interactions with SERCA at the University of Minnesota during the course of her Ph.D. These viewpoints have not been vetted with the law firm where she works or its clients, and do not represent the positions of the firm, its lawyers, or any of its clients. None of this writing is intended as legal advice and it should not be taken as such. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The inhibition was non-competitive, with Ki values of 2.34 μM, 0.455 μM and 0.342 μM under co-, pre- or pre/co-incubation conditions, respectively. Similar values were obtained for inhibition of [3H]MPP+ uptake by MATE1-overexpressing cells. Gefitinib, imatinib, pazopanib, sorafenib, and sunitinib also inhibited MATE1-mediated creatinine uptake. Further, all these TKIs except pazopanib inhibited [14C]creatinine uptake by OCT2-overexpressing cells. In rat kidney slices, the ratio of unbound tissue accumulation of TKIs to extracellular concentration ranged from 2.05 to 3.93. Prediction of the influence of TKIs on SCr based on the renal creatinine clearance and plasma maximum unbound concentrations of TKIs suggested that crizotinib and imatinib might increase SCr by more than 10% in the clinical context. Accordingly, it is necessary to be cautious in diagnosing TKI-induced renal failure only on the basis of an increase of SCr.Creatinine is actively secreted across tubular epithelial cells via organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 (MATE1). We previously showed that the tyrosine kinase inhibitor (TKI) crizotinib inhibits OCT2-mediated transport of creatinine. In the present work, we examined the inhibitory potency of TKIs, including crizotinib, on MATE1-mediated transport of creatinine. Then, we used the kinetic parameters estimated in this and the previous work to predict the potential impact of TKIs on serum creatinine level (SCr) via reversible inhibition of creatinine transport. Crizotinib inhibited [ In hospital practice, daily measurement of SCr is commonly used to monitor patients for AKI.Renal failure can be caused by various chemotherapeutic agents, and may require termination of the therapy or decrease of the dose. The Kidney Disease Improving Global Outcomes (KDIGO) clinical guidelines in 20122, which account for 10–40% of total creatinine clearance, depending on kidney function3. Creatinine is a substrate of organic cation transporter 1 (OCT1), OCT2, organic anion transporter 1 (OAT1), OAT2, OAT3, multidrug and toxin extrusion (MATE1), and MATE2K7. Genome-wide association studies indicate that genetic mutations of OCT2 and MATE1 affect creatinine clearance or SCr9, and OCT2 and MATE1 are expressed at the basolateral and apical membranes of renal tubular epithelial cells, respectively6. These creatinine transporters are inhibited by various drugs, including cimetidine, DX-619, pyrimethamine, and trimethoprim10. A physiologically based pharmacokinetic (PBPK) model analysis by Imamura et al. indicated that DX-619, an antibacterial agent, could reduce creatinine clearance by up to 51.4%, by decreasing the creatinine secretion mediated by these transporters10. Such an increase of SCr due to inhibition of creatinine transporters by drugs might lead to incorrect diagnosis of drug-induced renal failure. It is already known that TKIs such as crizotinib, gefitinib, imatinib, pazopanib, sorafenib, and sunitinib clinically increase SCr16, and have the potential to inhibit OCT2 and/or MATE120. Therefore, the increase of SCr by TKIs could be due to either true renal failure or reversible interaction with creatinine transporters. Thus, it is clinically important to evaluate the inhibitory potential of TKIs towards creatinine transporters in order to manage chemotherapy appropriately.Creatinine is mainly excreted into urine by glomerular filtration and is partly secreted via transporters20. Furthermore, the inhibitory effect of crizotinib on OCT2 was substrate-dependent. It is important to know whether inhibition of MATE1, which mediates renal secretion of creatinine at the apical membrane, by TKIs shows similar characteristics. Therefore, in the present study we examined on the inhibitory effects of TKIs, including crizotinib, on MATE1-mediated uptake of creatinine. Furthermore, we used the kinetic parameters obtained in the present and previous work to model the effect of TKI-induced inhibition of creatinine transporters on SCr in order to assess whether it might be significant in relation to the clinical diagnosis of TKI-induced renal failure.We recently reported that crizotinib is an inhibitor of OCT2-mediated uptake of creatinine, and we found that its inhibitory effect after pre-incubation or combined pre-incubation/co-incubation (pre/co-incubation) was greater than that in the case of co-incubation alone14C]creatinine by HEK293/MATE1 cells was significantly higher than that by mock cells, and since the uptake increased almost linearly up to 3 min . Then, we examined the time dependence of inhibition of MATE1-mediated [14C]creatinine uptake by crizotinib. We found that the extent of the inhibition increased time-dependently, reaching a plateau after 60 min pre-incubation  is due to intracellular accumulation of crizotinib. Accordingly, to test this idea, we examined the intracellular accumulation of crizotinib using monolayers of MDCKII/MATE1 cells in a Transwell system. Pre-incubation of the cells with crizotinib (2 μM) added on the basolateral side alone reduced 1Ccreatini14C]creatinine, the concentration dependence of creatinine uptake by HEK293/MATE1 and HEK293/OCT2 cells was examined in the presence of several TKIs. Firstly, in order to confirm that the pre-incubation effect is not specific to crizotinib, the effects of co- and pre/co-incubation with imatinib on [14C]creatinine uptake by HEK293/MATE1 cells were examined. Imatinib reduced [14C]creatinine uptake with IC50 values of 0.466 ± 0.053 μM and 0.0651 ± 0.0087 μM under co- and pre/co-incubation conditions, respectively  to 3.93 ± 0.15 (sunitinib), as shown in Table We also examined the tissue accumulation of crizotinib in rat kidney slices. The uptake of crizotinib at 37 °C was markedly higher than that at 4 °C -fused MATE1 protein in response to crizotinib. Accordingly, other mechanisms such as structural changes of MATE1 due to an allosteric effect of bound crizotinib, or dephosphorylation or degradation of MATE1, which could be associated with the pharmacological effect of crizotinib, should be considered. It has been reported that another TKI, dasatinib, reduced OCT2 activity by inhibiting the Src family kinase YES1, resulting in dephosphorylation of OCT219. Therefore, the mechanism by which crizotinib inhibits MATE1 might involve dephosphorylation of MATE1 via inhibition of an unknown kinase(s). In addition, we found that creatinine was not transported by MATE2K in the present study, contrary to a previous report6. This apparent discrepancy might be explained by differences in the amounts of MATE2K expressed in the two models. In any event, crizotinib was a markedly less potent inhibitor of MATE2K than MATE1. Therefore, MATE2K was not incorporated in the equation for prediction of creatinine clearance, as its contribution to the effect of TKIs is presumed to be small.Firstly, we considered the effect of pre-incubation on the inhibitory potential of crizotinib for MATE1-mediated transport of creatinine, because kidney tissues are clinically exposed to administered drugs for a long period of time and many previous studies have been done without pre-incubation. As shown in Fig. tes Fig. . The mecKp,uu, of crizotinib in kidney was estimated using rat kidney slices. For concentration setting, it is preferable to use the clinically relevant unbound concentration. However, the clinically unbound concentration of some TKIs is as low as about 10 nM. It is technically infeasible to use such low concentrations experimentally, because TKIs are extensively adsorbed on the experimental apparatus; therefore, the concentration of TKIs was unified to 1 μM in this study. In vitro uptake of crizotinib by the kidney slices showed a significant temperature dependence, suggesting that it is transporter-mediated, at least in part. Although the exact mechanism of crizotinib uptake into proximal tubular epithelial cells is unclear, it has been reported that several TKIs are substrates of solute carrier transporters, including OCT226. Thus, transporters expressed in tubular epithelial cells, such as OCT2 and organic anion transporter 3 (OAT3), may play a role in accumulation of TKIs in kidney. Nevertheless, it will be important in the future to establish the mechanism of the pre-incubation effect in order to ensure that the prediction of SCr change is soundly based.Next, to aid in predicting the inhibitory potential of TKIs on MATE1 in the clinical context, we estimated the intracellular concentrations of TKIs, because MATE1 excretes intracellular creatinine to the tubular lumen. Tissue accumulation, 10 and the reported Cmax,u, crizotinib could increase SCr by 14.5%, and the increment is increased to 25.3% when the Kp,uu value is taken into consideration. We did not incorporate MATE2K in the equation for prediction of SCr, because creatinine was negligibly transported by HEK293/MATE2K cells, and also because crizotinib was a much less potent inhibitor of MATE2K, compared with OCT2 and MATE1. Moreover, the intrinsic clearances for OCT2 and MATE1 are similar, since the protein amounts of OCT2 and MATE1 are comparable in human kidney27, and a previous study well predicted the clinical increase of SCr based on this assumption10. The clinically reported eGFR reduction in patients treated with crizotinib was 23.9% : 21.3–26.6%)28. Bearing in mind that crizotinib has a long half-life (t1/2) of 42 hr, the predicted increase of SCr estimated from Ctrough,u might also be consistent with the clinical observation. Our modeling analysis also showed that imatinib could increase SCr by 42.0% and 29.4% based on Cmax,u and Ctrough,u, respectively, and these values increased to 52.6% and 42.6%, respectively, after incorporation of Kp,uu. Again, these values are broadly consistent with the clinically reported decrease of eGFR of 27.1% . Considering a report that increasing effect of imatinib on SCr was reversible13, our conclusion here regarding imatinib appears to be clinically relevant. These results support the idea that specific interaction of some TKIs with renal creatinine transporters may lead to a reversible increase of SCr in the absence of renal failure. However, since it is also possible that crizotinib and imatinib cause true adverse events in kidney, we emphasize that it is desirable to use not only SCr, but also additional renal function markers such as blood urea nitrogen, to distinguish between reversible and true drug-induced renal failure. Moreover, it should be noted that incorporation of Kp,uu values in the prediction may or may not be necessary depending on the mechanism of the pre-incubation effect; it is necessary if the pre-incubation effect is caused by kinase inhibition, but not if the pre-incubation effect is determined by the intracellular concentration of drugs. Further study will be needed to clarify the mechanism in order to improve the prediction of the changes in creatinine disposition.Finally, we used the estimated kinetic parameters in a modeling analysis to investigate whether inhibition of transporters by TKIs might cause a clinically significant increase of SCr. As shown in Table In conclusion, the present study demonstrated that TKIs such as crizotinib and imatinib have the potential to inhibit creatinine transport via MATE1, as well as OCT2, at clinically relevant concentrations. The resulting reversible increase of SCr could lead to incorrect diagnosis of drug-induced renal failure.14C]Creatinine (58 mCi/mmol) and [3H]N-methyl-4-phenylpyridinium acetate  were purchased from Moravek Biochemicals  and American Radiolabeled Chemicals, Inc. , respectively. Non-labeled creatinine and crizotinib were purchased from Wako Pure Chemical Industries, Ltd.,  and AdooQ Bioscience, LLC , respectively. All other chemicals were commercial products of reagent grade. pCMV-SPORT6/MATE1 (clone name: IRAK013O20) was obtained from RIKEN BRC .creatinine or [3H]MPP+ in the absence or the presence of TKIs. The cells were incubated at 37 °C. Uptake was terminated by washing the cells three times with 0.5 ml of ice-cold TM, and the cells were solubilized in 0.25 ml of 0.01% (v/v) Triton-X. Radioactivity was measured with a liquid scintillation counter . Part of the lysate was used for the determination of total protein amount with a protein assay kit . For uptake assay using the Transwell system, MDCKII cells were plated at a density of 2.2 × 105 cells/cm2 on a 12-well Transwell filter insert for 5 days before uptake study. After adding TM containing 2 μM crizotinib for 60 min, the medium on both sides was removed and the cells were washed three times with ice-cold TM. Uptake study was initiated by adding TM containing [14C]creatinine and TM alone on the apical and basolateral sides, respectively. Uptake was terminated by washing the cells three times with ice-cold TM, and intracellular [14C]creatinine concentration was determined by the method described above. In the experiments with crizotinib, non-labeled creatinine was used and the concentration was determined by LC-MS/MS. After initiation of uptake, the medium on the apical side was collected at 2 min, and the crizotinib concentration was determined.The uptake experiment was conducted as described previously31. Whole kidneys of male rats were sliced (0.3 mm thick) with a microslicer . The slices were immediately put in ice-cold oxygenated transport buffer . Three or four slices per rat, each weighing 5 to 20 mg, were randomly selected and pre-incubated in a 12-well plate with 2.0 ml/well of oxygenated transport buffer at 37 °C for 5 min. After pre-incubation, the kidney slices were placed in transport buffer containing TKIs to initiate the uptake reaction. After uptake at 37 °C for an appropriate time, each slice was rapidly removed from the transport buffer, washed twice in ice-cold transport buffer, blotted on filter paper and weighed. Non-specific adsorption of each substrate was estimated as the absorption on a slice incubated at 4 °C for 1 min. The tissue accumulation of TKIs was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described below.Uptake study by rat kidney slices was carried out as described previously, with slight modificationsThe amounts of TKIs were determined with a LCMS-8050 triple quadrupole LC-MS/MS  coupled to an LC-30A system (Shimadzu) using an ACQUITY UPLC BEH C18 column  at 40 °C. The mobile phase was composed of a mixture of 0.1% formic acid in water (pH 3.0) and 0.1% formic acid in acetonitrile at the flow rate of 0.2 mL/min. The mass numbers of the molecular and product ions for each compound were as follows: crizotinib , gefitinib , imatinib  pazopanib , sorafenib , and sunitinib . Labsolutions software  was used for data manipulation. The detection limit was 10 nM for each compound.Experiments were conducted at least twice, and the results are shown as representative values in individual experiments. Uptake by transporter-expressing cells was expressed as the cell-to-medium (C/M) ratio (μL/mg protein), obtained by dividing the uptake amount by the concentration of substrate in the TM. MATE1- or OCT2-mediated uptake was obtained after subtraction of the uptake by mock cells.IC50 value was obtained by means of the following Eq. , and are shown as mean +/− statistical errors from a single experiment. The inhibitory effect of TKIs was expressed as a percentage of the control, and the wing Eq. ;1\documev, Vmax, Km, and [C] are initial uptake rate, maximal uptake rate, Michaelis constant, and substrate concentration, respectively.Kinetic parameters for uptake were obtained using the Michaelis-Menten Eq. ;2\documeKi of crizotinib was obtained from the following Eq.  based on the steady-state uptake at 37 °C and 4 °C was defined according to Eq. (32;The unbound renal proximal tubule epithelial cell-to-medium concentration ratio (ss was calculated by subtracting bound compound after uptake for 1 min at 4 °C from the uptake for 120 min at 37 °C or 4 °C by rat kidney slices. Kp,uu was calculated by dividing The C/M ratio10;cre, CLcre′ FR, RPF, and CLrs,int,all represent creatinine clearance, CLcre in the presence of creatinine transporter inhibitor, the fraction reabsorbed, renal plasma flow, and the intrinsic clearance by tubular secretion, respectively. In this model, the intrinsic clearances of OCT2 and MATE1 were assumed to be equal. When MATE1 inhibition was predicted from the Cmax,u of drugs, Kp,uu was assumed to be 1. For the prediction of CLcre change, RPF was set as 545 mL/min/1.73 m2 according to Berg et al.33, and individual parameters CLcre, FR, GFR, and CLrs,int,all of 11 subjects were taken from the reported clinical study10.Model-based increase of SCr by TKIs was estimated according to Eqs  and 6 10SUPPLEMENTARY INFORMATION


However, the gating mechanisms of the Panx3 channels remain unclear. Here, we show that the Panx3 ER Ca2+ channel is modulated by phosphorylation of the serine 68 residue (Ser68) to promote osteoblast differentiation. Among the 17 candidate phosphorylation sites identified, the mutation of Ser68 to Ala (Ser68Ala) was sufficient to inhibit Panx3-mediated osteoblast differentiation via reduction of Osterix and ALP expression. Using a Ser68 phospho-specific antibody (P-Panx3) revealed Panx3 was phosphorylated in prehypertrophic, hypertrophic chondrocytes, and bone areas of the newborn growth plate. In osteogenic C2C12 cells, P-Panx3 was located on the ER membranes. Importantly, the Ser68Ala mutation only affected Panx3 ER Ca2+ channel function. Ser68 on Panx3 was phosphorylated by ATP stimulation and PI3K/Akt signaling. Finally, real-time FRET imaging and ratio analysis revealed that the Panx3 channel conformation was sensitive to ATP. Together, the phosphorylation of Panx3 at Ser68 is an essential step controlling the gating of the Panx3 ER Ca2+ channel to promote osteogenesis.Pannexin 3 (Panx3) is a regulator of bone formation. Panx3 forms three distinct functional channels: hemichannels, gap junctions, and endoplasmic reticulum (ER) Ca Intracellular Ca2+ levels ([Ca2+]i) is controlled by the extracellular space and the ER, a Ca2+ storage organelle. Ca2+ is released from the ER through ER Ca2+ channels located on the ER membrane. These receptors include the inositol-3-trisphosphate (IP3) receptors (IP3Rs) that are ubiquitously expressed2 and the ryanodine receptors (RyRs) expressed in some tissues3. Additionally, pannexins (Panxs), which are members of the gap junction protein family, were recently reported to act as ER Ca2+ channels. The Panx1 ER Ca2+ channel functions in prostate cancer cells4, and Panx3 acts as an ER Ca2+ channel in osteoblasts5.Intracellular Ca9. Vertebrates have two gap junction protein families: the connexins (Cxs) and the pannexins (Panxs). The Cxs have more than 20 members and their functions have been reported10. Dysregulations and mutations of Cxs are involved in many human diseases, including cancer, hypertension, atherosclerosis, and developmental abnormalities11. By contrast, Panxs are less well characterized and they consist of only three known members (Panx1–3)14. Panx1 is widely expressed, especially in the central nervous system, Panx2 is frequently expressed in the central nervous system15, and Panx3 expression has been reported in the skin, cochlea, and developing hard tissues, including cartilage, bones, and teeth20. The human disorders linked to the Panxs, and especially Panx1, are associated with ischemia, stroke, overactive bladder, HIV infections, Crohn’s disease, and platelet aggregation21. Further, a germline variant in PANX1 has been associated with dysfunctions that included intellectual disabilities, hearing loss, and other multisystem failures22. Panx3 has been linked to osteoarthritis (OA), a disabling degenerative joint disorder with cartilage destruction, subchondral bone remodeling, and inflammation of the synovial membrane23.Gap junctions regulate various intracellular and physiological functions via the exchange of small molecules, such as ATP and ions, from the cell to the extracellular space via hemichannels and from cell to cell via gap junctions24. Previously, we have identified that Panx3 promotes chondrocyte differentiation by the ATP released via the Panx3 hemichannel, which counteracts the parathyroid hormone (PTH)–related protein (PTHrP) signaling pathway16. We also reported that Panx3 promotes osteoblast differentiation via its functions as a hemichannel, an ER Ca2+ channel, and a gap junction5. In addition, Panx3 regulates the osteoprogenitor cell cycle exit by inhibiting Wnt/β-catenin signaling through its hemichannel25. Panx3-null (Panx3−/−) mice show low bone density and marked dwarfism caused by the failure in both endochondral and intramembranous ossification. An in vivo study showed that Panx3 regulates mature hypertrophic chondrocyte differentiation and is requred in osteogenesis from the early stage, whereas Cx43 plays a role in the maturation stage. We also demonstrated that Panx3 and Cx43 play distinct roles in bone formation26.Panx3 is now recognized as a new regulator of bone growth27. The tetramer of the subunit forms a channel structure that functions as a hemichannel, gap junction, and ER Ca2+ channel, and the ER Ca2+ channel is Panxs specific. Recently, Panx1 and Panx3 were recognized as N-linked glycosylate proteins. Panx1 has the glycosylation site at asparagine 254 in the second extracellular loop, on the other hand, Panx3 at asparagine 71 in the first extracellular loop28. Panx2 also contains a potential N-linked glycosylation consensus site at asparagine 86, although the glycosylation of this residue has not yet been confirmed. Glycosylation of Panxs plays a role in the appropriate trafficking of these Panxs to the cell surface29. However, the mechanisms controling the opening or closing of Panxs, and especially the Panx3 channel, are not yet understood.Both Cxs and Panxs have common protein structures, including four transmembrane domains, two extracellular loops, one intracellular loop, and N- and C-terminal segments2+ channel is activated by phosphorylation at the Ser68 residue by ATP-mediated PI3K/Akt signaling to promote osteoblast differentiation. Phosphorylation of Panx3 at Ser68 increases intracellular Ca2+ levels through Panx3 ER Ca2+ channel gating, but not via its hemichannel or gap junction functions. Our results reveal that the Panx3 ER Ca2+ channel is regulated by a distinct gating mechanism that differs from the mechanism regulating the hemichannel and gap junction functions.In this study, we showed that the Panx3 ER Ca31, and immunoprecipitation assays (IP). Pro-Q diamond phosphoprotein gel-staining methods were used: total Panx3 protein was immunoprecipitated with V5 antibody, followed by detection of phosphorylation with the Pro-Q gel-staining method. In both Pro-Q staining and IP, total Panx3 protein in immunoprecipitated cell lysate was detected by Western blot using V5 antibody. The amount of total extracted protein (Input) was confirmed with α-tubulin antibody. Cell lysates from the Panx3 overexpressing cells showed a phosphorylated band similar in size to the Panx3 molecular weight, 47 kD, after Pro-Q staining  to analyze the candidate sites. We found 17 phosphorylation candidates and made point mutation constructs of each Panx3 phosphorylation candidate site Fig. . We thenin vitro at Ser68. The protein lysate from Panx3 expressing C2C12 cells showed the expected band of P-Panx3 between 45 and 50 kD molecular weight, and the size of that band was diminished by CIP treatment and in cell lysates from Ser68Ala transfected cells  and tested its specificity with an ELISA assay. The P-Panx3 antibody specifically bound the antigen peptide (P-peptide) Fig. . We thenlls Fig. . These rlls Fig. . We immulls Fig. .Figure 3The next question we addressed was how P-Panx3 regulates Panx3 channel functions. We first determined the cellular location of P-Panx3 expression by immunocytochemistry. The merged image obtained with P-Panx3 and ER-tracker or Panx3 revealed that P-Panx3 was expressed strongly on ER membranes, rather than on the plasma membrane or gap junction. Quantitative analysis revealed that 75% of the P-Panx3 protein was colocalized with ER tracker, while no P-Panx3 expression was observed in the Ser68Ala stably transfected C2C12 cells Fig. . Further2+ channel function. We analyzed Ca2+ efflux from the ER and stimulation of Ca2+ storage by ATP. The Ser68Ala mutation inhibited Panx3-mediated Ca2+ efflux  analysis to examine the changes in Panx3 protein conformation by phosphorylation to P-Panx3. We created recombinant Panx3 or Ser68Ala with CFP to the N-terminus and YFP to the C-terminus. The FRET real-time imaging of cells co-transfected with CFP and YFP vectors showed that Panx3-expressing C2C12 cells showed FRET after ATP stimulation, while cells treated with apyrase or Ser68Ala expressing cells did not Fig. –6. Quant2+ channel is regulated by ATP/PI3K/Akt mediated phosphorylation at the Ser68 residue. P-Panx3 has a pronounced involvement in osteoblast differentiation but not in osteoprogenitor proliferation. P-Panx3 is localized on the ER membrane, and mutation of Ser68 inhibits Ca2+ efflux from Panx3 ER Ca2+ channel, but does not affect the ATP releasing hemichannel or the gap junction function, with the end result of propagation of intracellular Ca2+ to neighboring cells. The Panx3 hemichannel releases intracellular ATP to the extracellular space to promote the activation of the PI3K/Akt signaling pathway through purinergic receptors. Panx3 then undergoes phosphorylation at Ser68 on the ER membrane  may form a complex with Akt to activate the Panx3 ER Ca2+ channel. We recently found that Panx3 regulates proliferation and differentiation of odontoblasts by AMPK activity through ATP releasing hemichannel activities17. AMPKα1 and AMPKα2 KO mice are viable, but AMPKα1/α2 double KO mice show embryonic lethality41. The AMPKα1 KO mice have severely low bone mass, with both cortical and trabecular bone compartments being smaller43. Thus, AMPK could be involved in Panx3 channel regulation. Another potential candidate is BMP2 signaling, because it regulates Panx3 expression and activation of the Panx3 signaling pathway25. Thus, BMP2 may contribute to the activation of Panx3 channel. In present study, BMP2 was added for cell culture conditioning in several differentiation experiments. The results suggest that BMP2 may regulate Panx3 channel activation by phosphorylation. BMP2 signaling has two main pathways—smad dependent or independent44. Thus, smad-dependent signaling and an independent pathway, such as MAPK signaling, may interact to cause Panx3 ER Ca2+ channel activation.Several kinases are reported to bind and activate Akt2+ channel types would differ. In our screening with the ALP assay, we found another interesting site, Ser303 49. Cx43 is also well known as a phospho-substrate for mitogen-activated protein (MAP) kinases, as well as protein kinase C (PKC)52. The phosphorylation of Cx43 at the S368 residue reduces hemichannel activity53. Cx43 is also reported to be phosphorylated by Akt to traffic and stabilize the Cx43 protein in the plasma membrane54. Our present findings would allow the addition of a distinct regulation mechanism between Panx3 and Cx43. Although many molecules, including kinases, activate Cx43 channels, Akt works in different systems to control Panx3 and Cx43 channel gating.The gap junction proteins that are known to regulate bone formation are Panx3 and Cx43. These two proteins have their own distinct functions and expression patterns that function in their contribution to bone growth. Panx3 functions as an ER Ca2+ channel was phosphorylated at Ser68 residue by ATP/PI3K/Akt signaling. This phosphorylation mechanism is critical for promoting osteoblast differentiation. Our results reveal that the three Panx3 channels each have different activation mechanisms and function together to orchestrate osteogenesis.In summary, we have shown that the Panx3 ER Ca16. The Panx3 expression vector (pEF1/Panx3) and the control vector (pEF1) have been described previously16. In brief, the pEF1/Panx3 vector was constructed by cloning the coding sequence of mouse Panx3 cDNA into the pEF1/V5-His vector (Invitrogen). The antibodies were obtained for Ocn from Biomedical Technology; CaMKII and P-CaMKII from Cell Signaling Technology; P-NFATc1 from Santa Cruz Biotechnology, Inc.; V5 from Invitrogen; and NFATc1 from BD. HA was from COVANCE; α-tubulin from Sigma-Aldrich, CIP from New England Biolabs; Apyrase from Sigma-Aldrich; LY294002 from Invitrogen; BMP2 from Humanzyme; and iQ SYBR Green Supermix from Bio-Rad Laboratories. ER-tracker was obtained from Invitrogen. HRP-conjugated goat anti–mouse and goat anti–rabbit IgG were obtained from United States Biological. The Akt-CA was obtained from Addgene. The inhibitory Panx3 peptide was described previously16.A rabbit polyclonal P-Panx3 antibody was raised against a synthetic peptide  from the first extracellular loop of the mouse Panx3 protein and the native peptide was used as a control sequence (SCFSPSNFSC). Rabbit anti-Panx3 antibody was used as previously described2. For the experiments, C2C12 cells were induced from osteogenic cells by the addition of 50 µg/ml ascorbic acid (Sigma-Aldrich) and 5 mM β-glycerophosphate (Sigma-Aldrich). For the proliferation assay, the cells (2.5 × 103 cells/ml) were stably transfected with either pEF1/Panx3 or Ser68Ala or control pEF1 and cultured in 96-well plates for 2 days. The cell proliferation activity was evaluated using a cell counting kit (Dojindo). The absorbance was measured using a microplate reader. For osteoblast differentiation, transiently or stably transfected C2C12 cells (∼90% confluence) were cultured in the presence of 300 ng/ml BMP2 (Humanzyme) and 2% FBS. Primary calvarial cells were prepared from the calvaria of newborn mice and cultured in α-minimum essential medium  with 10% FBS, 100 units/ml of penicillin, and 100 µg/ml of streptomycin, as previously described55. For the experiment with primary calvarial cells, cells were induced by osteogenesis with 50 µg/ml ascorbic acid (Sigma-Aldrich) and 5 mM β-glycerophosphate (Sigma-Aldrich). All experimental procedures were approved by the Animal Care and Use Committee of the National Institute of Dental and Craniofacial Research (protocol no. 15-758) and Tohoku University (protocol no. 2017DnA-035). And all methods were carried out in accordance with relevant laws and legislations of both NIDCR and Tohoku Univeristy.C2C12 cells were grown in DMEM (Invitrogen) containing 10% FBS (HyClone) at 37 °C under 5% COProtein lysates isolated using PhosphoSafe extraction buffer (EMD Millipore) were run on SDS-PAGE. The phosphorylation band was analyzed by staining with Pro-Q Diamond phosphoprotein gel stain kit (Invitrogen) following the company’s standard protocol. Briefly, immunoprecipitated cell lysate by V5 antibody was loaded into NuPAGE Bis-Tris gels (Thermo). The gel was immersed in a fixed solution (50% methanol and 10% acetic acid) and then incubated at room temperature with gentle shaking for 1 hour. After washing, the gel was stained with Pro-Q Diamond phosphoprotein gel stain, with gentle agitation in the dark for 90 minutes. To reduce the background, the gel then was destained by incubating the gel in a destaining solution (Thermo) with gentle shaking for 30 minutes at room temperature, protected from light. Detaining was repeated several times until the background was reduced.Cytokines were measured by conventional ELISA  or multiplex ELISA (Quansys Biosciences). Both methods were performed as recommended by the manufacturer. If appropriate, cytokine data were normalized to total protein data  with the following formula: raw cytokine data (in pg/ml) / total protein concentration in the lysate (in mg/ml).The phosphorylation sites of interest in Panx3 were screened by constructing a mutation vector of each of 17 phosphorylation candidate residues using a QuikChange XL Site-Directed Mutagenesis Kit (Agilent Technologies). Each mutation construct was transiently transfected into C2C12 cells with Nucleofector (Lonza). The transfected cells were then plated into 96-well culture plates and grown to 100% confluence. The cells were then induced to differentiate into osteoblasts by addition of 300 ng/ml BMP2. The ALP activity was measured in cell layers using a p-nitrophenyl phosphate substrate and an incubation temperature of 37 °C, or it was determined by the tartrate-resistant acid phosphatase (TRACP) & ALP double-stain kit (Takara Bio Inc.). The protein concentration was determined by the BCA protein assay method (Thermo Fisher Scientific).For immunostaining of newborn growth plates, the tissues were fixed overnight in paraformaldehyde, embedded in paraffin, and cut into 10-µm sections. After deparaffinization and rehydration, the sections were processed for heat-induced epitope retrieval in pH 6.0 citrate buffer (Dako). The cultured cells were stained by first incubating them in ER-Tracker Red (Invitrogen) for 20 min, followed by fixation in acetone at −20 °C for 2 min. The cells were blocked with Power block  and reacted for 2 h at room temperature with primary antibodies. The primary antibodies were detected by Alexa 488 (Invitrogen) or Alexa 594 (Invitrogen) or by Cy-5–conjugated (Jackson ImmunoResearch Laboratories) secondary antibodies. Nuclear staining was performed using Hoechst dye (Sigma-Aldrich). The analysis was performed on an LSM 780 inverted confocal microscope .For immunolocalization of P-Panx3 protein, glutaraldehyde-fixed, Lowicryl-embedded thin sections were first reacted with the primary antibody diluted in PBS with 1% BSA and 0.1% Triton X-100 (1/100 diluted anti-P-Panx3) for 1 h at room temperature. After washing in PBS, grids were incubated for 30 min at room temperature with the goat 1/25 diluted anti-rabbit secondary antibody (Nanoprobes) coupled to 10 nm gold particles diluted in PBS. Following final washing in PBS, the grids were rapidly rinsed in a jet of distilled water, air-dried, and counterstained with 4% uranyl acetate. The sections were analyzed with a Hitachi H7600 transmission electron microscope.16. C2C12 cells stably expressing pEF1, pEF1/Panx3, or Ser68Ala were seeded at 1.0 × 104 cells/well in a 96-well plate, and cultured for 2 days in DMEM containing 10% FBS. The cells were then washed with PBS, followed by incubation in PBS for 2 min. The supernatant was collected and assayed with luciferase and luciferin (Promega). The luminescence was measured using a Mithras LB 940 multimode plate reader (Berthold).The ATP flux was examined by luminometry, as previously described2+]i and imaging of Ca2+ wave propagation were examined as previously described5. The [Ca2+]i measurements were conducted on C2C12 cells stably expressing pEF1, pEF1/Panx3, or Ser68Ala cultured in a 96-well plate for 3 days cultured with DMEM containing 10% FBS and then loaded with 5 µM Fura-2AM (Invitrogen) in Ca2+ containing HBSS for 45 min at 37 °C in 5% CO2. The Ca2+ transients in Ca2+-free HBSS were recorded as the 340/380 nm ratio (R) of the resulting 510 nm emissions determined using a plate reader . For the stimulation, 200 µM of ATP was automatically injected into the cell cultures by the LB942. The Ca2+ wave propagation was imaged by seeding C2C12 cells stably expressing pEF1, pEF1/Pnax3, or Ser68Ala in a glass-bottomed dish and incubating in Ca2+ containing HBSS containing 4 µM Fluo-4 AM Ca2+ indicator (Invitrogen), 10 µM pluronic F-127 (Invitrogen), 0.1% OxyFluor (Oxyrase), and 2.5 µM caged reagent NP-EGTA AM (Invitrogen) for 30 min at room temperature, followed by washing and incubation with Ca2+-free HBSS. The Ca2+ was uncaged by a two-photon laser set at 730 nm focused on a single cell. The Ca2+ wave propagation was measured at 10 s after the illumination.The [Ca2 LB942; Berthold Technologies). 200 µM of ATP stimulation was auto-injected by the LB942.The plasmids encoding FRET biosensors were constructed by cloning the coding sequence of mouse normal or Ser68 mutated Panx3 cDNA into the pEYFP-N1 and pECFP-C1 vectors (Clontech). C2C12 cells transfected with biosensors were plated on glass-bottomed dishes and cultured with Phenol-Red-free DMEM containing 10% FBS. Time-lapse FRET imaging was performed on a Zeiss LSM 780 confocal microscope . The donor and transfer signals were excited by the 458 nm line of an Argon laser and detected simultaneously between 463–509 nm and 519–620 nm. Images were collected from the middle plane of cells. Time-lapse FRET imaging was captured for 30 s after ATP stimulation. To inhibit ATP receptors, the cells were incubated with 20|U Apyrase before capturing the images. To measure FRET ratio, FRET biosensor vectors including normal or Ser68 mutated Panx3 cDNA transiently transfected C2C12 cells were cultured in a 96-well for 2 days. The FRET ratio was recorded as the 485/536 nm ratio using a plate reader (TriStar16. A 10 µg sample of each protein was electrophoresed in 4–12% SDS-polyacrylamide gel (Invitrogen) and transferred onto a polyvinylidene difluoride membrane using an iBlot device (Invitrogen). The membranes were immunoblotted with antibodies using standard protocols.The cell lysates were prepared as previously describedEach experiment was repeated several times and the data were analyzed using Prism 5 software. Student’s t-tests were used to highlight differences between two groups of data. One-way ANOVA was used for the quantification of band density ratios Fig. , ALP actSupplementary informationSupplemental Movie 1Supplemental Movie 2Supplemental Movie 3Supplemental Movie 4Supplemental Movie 5Supplemental Movie 6


In this study, we identified another phenolic compound, methyl 4-hydroxycinnamate (MHC), which among several tested phytochemicals could uniquely cooperate with CA in killing AML cells, but not normal peripheral blood mononuclear cells. Notably, our data revealed striking phenotypical and mechanistic similarities in the apoptotic effects of MHC+CA and CUR+CA on AML cells. Yet, we show that MHC is a non-fluorescent molecule, which is an important technical advantage over CUR that can interfere in various fluorescence-based assays. Collectively, we demonstrated for the first time the antileukemic activity of MHC in combination with another phenolic compound. This type of synergistically acting combinations may represent prototypes for novel antileukemic therapy.Acute myeloid leukemia (AML) is a malignant hematopoietic disease with poor prognosis for most patients. Conventional chemotherapy has been the standard treatment approach for AML in the past 40 years with limited success. Although, several targeted drugs were recently approved, their long-term impact on survival of patients with AML is yet to be determined. Thus, it is still necessary to develop alternative therapeutic approaches for this disease. We have previously shown a marked synergistic anti-leukemic effect of two polyphenols, curcumin (CUR) and carnosic acid (CA), on AML cells  In the past four decades, there have been no major changes in the standard AML chemotherapy regimen. Although in 2017–2018 eight new promising AML drugs were approved by the U.S. Food and Drug Administration (FDA) Stone, , curcumiAllium cepa)  leaves  and CA. MHC is found in several plants, such as green onion , penicillin, streptomycin, and HEPES were purchased from Biological Industries . Stock solutions of curcumin (5 mM) and carnosic acid (10 mM) were prepared in absolute ethanol and methyl 4-hydroxycinnamate (50 mM) in DMSO.Curcumin (≥90%) and carnosic acid (98%) were purchased from Cayman Chemicals  and Chemlin UK , respectively. Methyl 4-hydroxycinnamate (96%) was synthesized by Dr. Katarzyna Sidoryk , as described previously  using an automatic trypan blue exclusion assay. The number of viable (trypan blue-impermeable) cells was counted directly, and cell viability was calculated as the percentage of viable cells relative to the total (viable + dead) cell count.Human AML cell lines, such as KG-1a stem-like cells (CCL-246.1) and HL60 myoblastic cells (CCL-240), were purchased from American Type Culture Collection . Cells were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and 10 mM HEPES (pH = 7.4) in a humidified atmosphere of 95% air and 5% COCells were collected by centrifugation and double stained with 14 μg/ml acridine orange and 14 μg/ml ethidium bromide, as described previously  and loaded with TMRE (100 nM), for 30 min in the dark, washed and resuspended in serum-free medium. Changes in mitochondrial membrane potential were assessed by flow cytometry.2+]cyt, cells were harvested, washed and incubated with 2.5 μM Fluo-3/AM in calcium (2 mM)-supplemented Ringer's solution  of several phytochemicals  for furt and store-operated Ca2+ channels (Since CUR+CA-induced apoptosis was previously found to be mediated by a sustained [Cachannels  (Dobrydnchannels . Likewischannels . The latchannels .CUR is known as a fluorescent molecule and is considered a pan-assay interference compound (PAINS) (Nelson et al., in vitro. This similarity was evidenced by a comparable ability of the two combinations to induce caspase- and calcium-dependent apoptosis (The major novel finding of this study is that, similar to CUR (Pesakhov et al., poptosis  which wapoptosis  or reducpoptosis . Interespoptosis . These fpoptosis . Apart fpoptosis  which mapoptosis . FurtherAll datasets generated for this study are included in the manuscript and/or the MD, AT, and KS conceived the concept. AT and MD designed experiments and wrote the manuscript. AT and SM performed experiments. KS and MC provided reagents.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We employed the photolabile Ca2+ chelator o‐nitrophenyl EGTA to induce bursts of intracellular free Ca2+ by laser photolysis in the epithelial tissue. Ca2+ bursts appear within seconds and are restricted to individual target cells. Cell contraction reliably followed within a minute, causing an approximately 50% drop in the cross‐sectional area. Increased Ca2+ levels are reversible, and the target cells further participated in tissue morphogenesis. Depending on Rho kinase (ROCK) activity but not RhoGEF2, cell contractions are paralleled with non‐muscle myosin II accumulation in the apico‐medial cortex, indicating that Ca2+ bursts trigger non‐muscle myosin II activation. Our approach can be, in principle, adapted to many experimental systems and species, as no specific genetic elements are required.The spatial and temporal dynamics of cell contractility plays a key role in tissue morphogenesis, wound healing, and cancer invasion. Here, we report a simple optochemical method to induce cell contractions  Drosophila embryos triggers a calcium burst and subsequent myosin‐mediated contraction of the apical surface of the cell.Uncaging EGTA with UV illumination in  Contractility underlies manifold processes in cell and tissue morphogenesis, including cell migration, cell shape changes, or junction collapse 2 leading to interference with phosphoinositol metabolism and subsequent suppression of cortical actin polymerization For controlling cell contractility, optogenetic approaches have recently been developed. Cell contractility can be inhibited by optically induced membrane recruitment of PIP2+) are known to be an important regulator of contractility in many cell types. Ca2+ plays a central role not only in muscle contraction, but also in cultured epithelial cells Drosophila oogenesis, tissue‐wide increase in intracellular Ca2+ activates myosin II and impairs egg chamber elongation Xenopus, a transient increase in Ca2+ concentration induces apical constriction in cells of the neural tube 2+‐induced contraction in non‐muscle cells remains to be resolved, it conceivably offers a simple and temporally precise way to interfere with and control contractile activity. In neuroscience, optochemical methods for the release of intracellular Ca2+ have been well established and widely employed 2+‐mediated light activation of myosin (CaLM) on the scale of seconds and at single‐cell resolution during tissue morphogenesis in Drosophila embryos. Optochemical control of contractility by Ca2+ uncaging has minimal spectral overlap with fluorescent protein reporters and optogenetic activators. Our results provide evidence for a ROCK‐dependent effect of increased intracellular Ca2+ on activating non‐muscle myosin II and its recruitment to the actomyosin cortex.Optochemical methods represent an alternative to genetically encoded sensor and effector proteins 2+ chelator o‐nitrophenyl EGTA (NP‐EGTA) 2+ concentration 2+ once the AM moiety is cleaved off by intracellular esterase. The o‐nitrophenyl EGTA‐Ca2+ complex cannot get out again because the AM moiety has been cleaved off by intracellular esterase. Following microinjection into staged embryos, uncaging was induced in the focal volume with a diameter of 2–3 μm and thus an area of 5 μm2 of a pulsed 355‐nm laser beam . The epidermis during this stage constitutes a columnar epithelium with a cell diameter in the range of about 8 μm and cell height of about 25 μm  showed a fourfold increase within 2‐s. Afterward, GCaMP6s fluorescence gradually decreased to near initial levels within a few minutes . We observed the typical contraction after the first pulse but no further obvious contractions after the second and third UV pulses Fig A and B. 2+‐induced cell contraction. Given their time scale in the minute range, it is unlikely that slow transcriptional or translational processes are involved. It is also unlikely that Ca2+ directly activates contraction similar to its role in muscle cells due to the distinct organization of cortical actomyosin and indicated by the substantial time lag between Ca2+ increase and cell contraction. Ca2+ may activate myosin II, similar to what has been reported for the Drosophila egg chamber 2+‐dependent protein kinases or phosphatases, such as myosin light‐chain kinase (MLK) Multiple mechanisms are conceivable for Ca2+‐induced cell contraction, we imaged myosin II dynamics following uncaging in embryos expressing E‐Cad‐GFP to label cell–cell contacts and sqh‐mCherry . sqh‐mCherry fluorescence is a direct indicator of active myosin II mini filaments, which are visible as clusters. Myosin II is found associated with adherens junctions  and at the apical cortex , where it is responsible for apical constriction As a first step toward identifying the mechanism of CaOne expects that a contracting cell applies a force on the junctional complexes linking it to its neighbors within the epithelium Fig A. To assDrosophila embryos 2+ uncaging, we therefore performed laser ablation on the first neighboring junctions of the CaLM‐activated contracting cell  (HindIII‐Xho1) and eGFP (EcoR1‐Xho1) were inserted between the EcoR1‐Xho1 sites of a pUASt with attB sequence. PCR cloning was verified by sequencing of the fragments. pUASt‐attB‐VinculinD1‐eGFP was inserted in chromosome II and recombined with E‐Cad‐mTomato. Homozygous lines were healthy and fertile.The Rho sensor is a bicistronic cassette that contains tdTomato fused to the Rho‐binding domain (RBD) from Anillin aa748–1,239) followed by a P2A peptide and membrane marker, tdKatushka2, fused to the CAAX box from human KRAS. The utility of the Anillin‐RBD for detecting regions of active Rho has been validated previously  followedEmbryos were prepared as previously described 2 (pH 7.2)] 2+ uncaging in epidermal cells, and 1 mM NP‐EGTA, AM was injected for Ca2+ uncaging in amnioserosa cells. To inhibit Rock activity, 10 mM Y‐27632 (Sigma) in water was injected.NP‐EGTA, AM (Invitrogen) was prepared in 1× injection solution [180 mM NaCl, 10 mM HEPES, 5 mM KCl, 1 mM MgCl2+ uncaging. The intensity of the UV laser was adjusted so that no morphological changes were induced in 1× injection solution‐injected embryos. The laser was applied for 1.5 s (around 300 pulses) per cell with 2.5% laser power (~ 0.5 mJ/cell).We employed a pulsed 355‐nm YAG laser  mounted on the epiport. We illuminated under the “Click and Fire” Mode on the “REO‐SysCon‐Zen” platform (Rapp OptoElectonic), while a movie was recorded via a spinning disk mounted on the side port . For the images in Figs 2+ sensor GCaMP6‐myr was maternally expressed with Mat‐Gal4‐67, 15  were recorded with a frame rate of 0.1/s on a spinning disk microscope  with an emCCD camera . The recording started about 20 s after Ca2+ uncaging.α‐Catenin‐RFP and apoptosensor were expressed under control of the driver AS‐Gal4 in the amnioserosa Fig A. Stage 2+ uncaging was applied during recording. In Fig EV4F, axial stacks of 11 images with 0.5 μm step size were recording from an embryo undergoing cellularization with an emCCD camera . In Fig EV4G, the cross‐sectional images were recorded in the GFP channel with a frame rate of 0.2/s from a stage 8 embryo with an emCCD camera .The Rho sensor was recorded in the GFP channel with a frame rate of 0.2/s Fig G and J. In Fig Embryos were fixed, stained, and mounted as previously described 2+ uncaging was applied during recording. After the target cell started to contract, the 1st neighboring junction was ablated with the 10% of laser power, and 200 ms (around 40 pulses) exposure time during the recording mode   with a CCD camera. Ca1.4) Fig F. The coIt. The background Ib was determined from the integrated density (a.u.), which was measured from the cytoplasm and divided by the measurement length (μm). The normalized GCaMP6‐myr intensity fold increase was calculated as follows:It is the mean intensity at time t, Ib is the mean intensity of the background at time t, I1− is the mean intensity at 1‐s before UV illumination, and I1b− is the mean intensity of the background at 1 s before UV illumination.The fluorescence intensity of GCaMP6‐myr Figs  and 6D w2+ uncaging, image stacks were projected by the “Max Intensity” option. The projected and cross‐sectional images were segmented and tracked with “Tissue Analyzer” To analyze cell dynamics after Ca2+ uncaging .To analyze myosin dynamics after Caging Fig , the imaIt. The background Ibt represents the averaged fluorescence intensity (a.u.) within the cytoplasm. The normalized Rho sensor intensity was calculated as follows: I = It/Ibt.To analyze Rho sensor dynamics, the fluorescence intensity of Rho sensor Fig J was meat = 0). To analyze VinculinD1 and E‐Cadherin dynamics, the fluorescence intensity of VinculinD1‐GFP .The ratio of VincuinD1‐GFP/E‐cadherin‐mTomato Fig C was genP in Fig B at cellDK conducted the experiments and analyzed the data. ZL generated the VinculinD1‐GFP transgenic fly and analyzed the VinculinD1‐GFP data. MH analyzed data and obtained in Figs The authors declare that they have no conflict of interest.Expanded View Figures PDFClick here for additional data file.Movie EV1Click here for additional data file.Movie EV2Click here for additional data file.Movie EV3Click here for additional data file.Movie EV4Click here for additional data file.Movie EV5Click here for additional data file.Movie EV6Click here for additional data file.Movie EV7Click here for additional data file.Movie EV8Click here for additional data file.Movie EV9Click here for additional data file.Review Process FileClick here for additional data file.