Rcn1, the fission yeast homolog of human DSCR1, regulates arsenite tolerance independently from calcineurin.

Calcineurin (CN) is a conserved Ca2+/calmodulin-dependent phosphoprotein phosphatase that plays a key role in Ca2+ signaling. Regulator of calcineurin 1 (RCAN1), also known as Down syndrome critical region gene 1 (DSCR1), interacts with calcineurin and inhibits calcineurin-dependent signaling in various organisms. Ppb1, the fission yeast calcineurin regulates Cl--homeostasis, and Ppb1 deletion induces MgCl2 hypersensitivity. Here, we characterize the conserved and novel roles of the fission yeast RCAN1 homolog rcn1+. Consistent with its role as an endogenous calcineurin inhibitor, Rcn1 overproduction reproduced the calcineurin-null phenotypes, including MgCl2 hypersensitivity and inhibition of calcineurin signaling upon extracellular Ca2+ stimuli as evaluated by the nuclear translocation and transcriptional activation of the calcineurin substrate Prz1. Notably, overexpression of rcn1+ causes hypersensitivity to arsenite, whereas calcineurin deletion induces arsenite tolerance, showing a phenotypic discrepancy between Rcn1 overexpression and calcineurin deletion. Importantly, although Rcn1 deletion induces modest sensitivities to arsenite and MgCl2 in wild-type cells, the arsenite tolerance, but not MgCl2 sensitivity, associated with Ppb1 deletion was markedly suppressed by Rcn1 deletion. Collectively, our findings reveal a previously unrecognized functional collaboration between Rcn1 and calcineurin, wherein Rcn1 not only negatively regulates calcineurin in the Cl- homeostasis, but also Rcn1 mediates calcineurin signaling to modulate arsenite cytotoxicity.

Systematic integration of molecular and clinical approaches in HCV-induced hepatocellular carcinoma.

BACKGROUND: MicroRNAs (miRNAs) play a crucial role in gene expression and regulation, with dysregulation of miRNA function linked to various diseases, including hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). There is still a gap in understanding the regulatory relationship between miRNAs and mRNAs in HCV-HCC. This study aimed to investigate the function and effects of persistent HCV-induced miRNA expression on gene regulation in HCC. METHODS: MiRNA array data were used to identify differentially expressed miRNAs and their targets, and miRNAs were analyzed via DIANA for KEGG pathways, gene ontology (GO) functional enrichment, and Ingenuity Pathways Analysis (IPA) for hepatotoxicity, canonical pathways, associated network functions, and interactive networks. RESULTS: Seventeen miRNAs in L-HCV and 9 miRNAs in S-HCV were differentially expressed, and 5 miRNAs in L-HCV and 5 miRNAs in S-HCV were significantly expressed in liver hepatocellular carcinoma (LIHC) tumors. Grouped miRNA survival analysis showed that L-HCV miRNAs were associated with survival in LIHC, and miRNA‒mRNA targets regulated viral carcinogenesis and cell cycle alteration through cancer pathways in LIHC. MiRNA-regulated RCN1 was suppressed through miRNA-oncogene interactions, and suppression of RCN1 inhibited invasion and migration in HCC. CONCLUSION: Persistent HCV infection induced the expression of miRNAs that act as tumor suppressors by inhibiting oncogenes in HCC. RCN1 was suppressed while miRNAs were upregulated, demonstrating an inverse relationship. Therefore, hsa-miR-215-5p, hsa-miR-10b-5p, hsa-let-7a-5p and their target RCN1 may be ideal biomarkers for monitoring HCV-HCC progression.

Loss of parkin causes endoplasmic reticulum calcium dyshomeostasis by upregulation of reticulocalbin 1.

Increasing evidence suggests that astrocytes play an important role in the progression of Parkinson's disease (PD). Previous studies on our parkin knockout mouse demonstrated a higher accumulation of damaged mitochondria in astrocytes than in surrounding dopaminergic (DA) neurons, suggesting that Parkin plays a crucial role regarding their interaction during PD pathogenesis. In the current study, we examined primary mesencephalic astrocytes and neurons in a direct co-culture system and discovered that the parkin deletion causes an impaired differentiation of mesencephalic neurons. This effect required the parkin mutation in astrocytes as well as in neurons. In Valinomycin-treated parkin-deficient astrocytes, ubiquitination of Mitofusin 2 was abolished, whereas there was no significant degradation of the outer mitochondrial membrane protein Tom70. This result may explain the accumulation of damaged mitochondria in parkin-deficient astrocytes. We examined differential gene expression in the substantia nigra region of our parkin-KO mouse by RNA sequencing and identified an upregulation of the endoplasmic reticulum (ER) Ca2+ -binding protein reticulocalbin 1 (RCN1) expression, which was validated using qPCR. Immunostaining of the SN brain region revealed RCN1 expression mainly in astrocytes. Our subcellular fractionation of brain extract has shown that RCN1 is located in the ER and in mitochondria-associated membranes (MAM). Moreover, a loss of Parkin function reduced ATP-stimulated calcium-release in ER mesencephalic astrocytes that could be attenuated by siRNA-mediated RCN1 knockdown. Our results indicate that RCN1 plays an important role in ER-associated calcium dyshomeostasis caused by the loss of Parkin function in mesencephalic astrocytes, thereby highlighting the relevance of astrocyte function in PD pathomechanisms.

The role of protein phosphatase 2A (PP2A) in the unfolded protein response (UPR) of plants.

Protein phosphatase 2A (PP2A) is a key regulator of plant growth and development, but its role in the endoplasmic reticulum (ER) stress response remains elusive. In this study, we investigated the function of PP2A under ER stress using loss-of-function mutants of ROOTS CURL of NAPHTHYLPHTHALAMIC ACID1 (RCN1), a regulatory A1 subunit isoform of Arabidopsis PP2A. RCN1 mutants (rcn1-1 and rcn1-2) exhibited reduced sensitivity to tunicamycin (TM), an inhibitor of N-linked glycosylation and inducer of unfolded protein response (UPR) gene expression, resulting in less severe effects compared to wild-type plants (Ws-2 and Col-0). TM negatively impacted PP2A activity in Col-0 plants but did not significantly affect rcn1-2 plants. Additionally, TM treatment did not influence the transcription levels of the PP2AA1(RCN1), 2, and 3 genes in Col-0 plants. Cantharidin, a PP2A inhibitor, exacerbated growth defects in rcn1 plants and alleviated TM-induced growth inhibition in Ws-2 and Col-0 plants. Furthermore, cantharidin treatment mitigated TM hypersensitivity in ire1a&b and bzip28&60 mutants. These findings suggest that PP2A activity is essential for an efficient UPR in Arabidopsis.

Reticulocalbin 1 is required for proliferation and migration of non-small cell lung cancer cells regulated by osteoblast-conditioned medium.

Reticulocalbin1 (RCN1) is implicated in tumorigenesis and tumour progression. However, whether RCN1-mediated bone metastasis of non-small cell lung cancer (NSCLC) cells was elusive. Here, we assessed the effect of osteoblast-conditioned medium (CM) on proliferation and migration of NSCLC cell line, NCI-H1299 and NCI-H460 cells, and identified the soluble mediators in CMs from osteoblasts and NSCLC cells using MTT, Clonogenicity, Transwell, wound healing, RT-PCR, and Western blotting assays, and LC-MS/MS analysis, respectively. Furthermore, the role of RCN1 was investigated in NSCLC cells cultured with or without osteoblast-CM. Tumour growth and bone resorption were measured in a nude mouse model bearing NCI-H1299 cells transduced with shRNA/RCN1 vector using in vivo imaging technique and micro-CT. The results showed that RCN1 with a higher abundance in osteoblast-CM, which was present in extracellular vesicles (EVs), enhanced RCN1 expression in NSCLC cells. Osteoblast-CM partially offset the inhibitory effect of RCN1 depletion on proliferation and migration of NSCLC cells. RCN1 depletion-induced endoplasmic reticulum (ER) stress caused by increasing GRP78, CHOP, IRE1α, p-IRE1α, p-PERK and p-JNK, which was positively regulated by self-induced autophagy, contributed to suppression of proliferation and migration in NCI-H1299 cells. Therefore, osteoblasts produced RCN1 to transfer into NSCLC cells partially through EVs, facilitating proliferation and migration of NSCLC cells via blocking ER stress. RCN1 could be required for proliferation and migration of NSCLC cells regulated by osteoblast-CM.

The OsCYP19-4 Gene Is Expressed as Multiple Alternatively Spliced Transcripts Encoding Isoforms with Distinct Cellular Localizations and PPIase Activities under Cold Stress.

Alternative splicing (AS) is an important molecular mechanism by which single genes can generate multiple mRNA isoforms. We reported previously that, in Oryza sativa, the cyclophilin 19-4 (OsCYP19-4.1) transcript was significantly upregulated in response to cold stress, and that transgenic plants were cold tolerant. Here we show that, under cold stress, OsCYP19-4 produces eight transcript variants by intron retention and exon skipping, resulting in production of four distinct protein isoforms. The OsCYP19-4 AS isoforms exhibited different cellular localizations in the epidermal cells: in contrast to OsCYP19-4.1, the OsCYP19-4.2 and OsCYP19-4.3 proteins were primarily targeted to guard and subsidiary cells, whereas OsCYP19-4.5, which consists largely of an endoplasmic reticulum (ER) targeting signal, was co-localized with the RFP-BiP marker in the ER. In OsCYP19-4.2, the key residues of the PPIase domain are altered; consistent with this, recombinant OsCYP19-4.2 had significantly lower PPIase activity than OsCYP19-4.1 in vitro. Specific protein-protein interactions between OsCYP19-4.2/3 and AtRCN1 were verified in yeast two-hybrid (Y2H) and bimolecular fluoresence complementation (BiFC assays), although the OsCYP19-4 isoforms could not bind each other. Based on these results, we propose that two OsCYP19-4 AS isoforms, OsCYP19-4.2 and OsCYP19-4.3, play roles linking auxin transport and cold stress via interactions with RCN1.

RCN1/OsABCG5, an ATP-binding cassette (ABC) transporter, is required for hypodermal suberization of roots in rice (Oryza sativa).

Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP-binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP-RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C(28) and C(30) fatty acids or ω-OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier.

Downregulation of RCN1 promotes pyroptosis in acute myeloid leukemia cells.

Reticulocalbin-1 (RCN1) is expressed aberrantly and at a high level in various tumors, including acute myeloid leukemia (AML), yet its impact on AML remains unclear. In this study, we demonstrate that RCN1 knockdown significantly suppresses the viability of bone marrow mononuclear cells (BMMNCs) from AML patients but does not affect the viability of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSCs) from healthy donors in vitro. Downregulation of RCN1 also reduces the viability of AML cell lines. Further studies showed that the RCN1 knockdown upregulates type I interferon (IFN-1) expression and promotes AML cell pyroptosis through caspase-1 and gasdermin D (GSDMD) signaling. Deletion of the mouse Rcn1 gene inhibits the viability of mouse AML cell lines but not the hematopoiesis of mouse bone marrow. In addition, RCN1 downregulation in human AML cells significantly inhibited tumor growth in the NSG mouse xenograft model. Taken together, our results suggest that RCN1 may be a potential target for AML therapy.

EGFRvIII Promotes Cell Survival during Endoplasmic Reticulum Stress through a Reticulocalbin 1-Dependent Mechanism.

Reticulocalbin 1 (RCN1) is an endoplasmic reticulum (ER)-residing protein, involved in promoting cell survival during pathophysiological conditions that lead to ER stress. However, the key upstream receptor tyrosine kinase that regulates RCN1 expression and its potential role in cell survival in the glioblastoma setting have not been determined. Here, we demonstrate that RCN1 expression significantly correlates with poor glioblastoma patient survival. We also demonstrate that glioblastoma cells with expression of EGFRvIII receptor also have high RCN1 expression. Over-expression of wildtype EGFR also correlated with high RCN1 expression, suggesting that EGFR and EGFRvIII regulate RCN1 expression. Importantly, cells that expressed EGFRvIII and subsequently showed high RCN1 expression displayed greater cell viability under ER stress compared to EGFRvIII negative glioblastoma cells. Consistently, we also demonstrated that RCN1 knockdown reduced cell viability and exogenous introduction of RCN1 enhanced cell viability following induction of ER stress. Mechanistically, we demonstrate that the EGFRvIII-RCN1-driven increase in cell survival is due to the inactivation of the ER stress markers ATF4 and ATF6, maintained expression of the anti-apoptotic protein Bcl-2 and reduced activity of caspase 3/7. Our current findings identify that EGFRvIII regulates RCN1 expression and that this novel association promotes cell survival in glioblastoma cells during ER stress.

Integrative Analyses and Verification of the Expression and Prognostic Significance for RCN1 in Glioblastoma Multiforme.

Glioblastoma multiform is a lethal primary brain tumor derived from astrocytic, with a poor prognosis in adults. Reticulocalbin-1 (RCN1) is a calcium-binding protein, dysregulation of which contributes to tumorigenesis and progression in various cancers. The present study aimed to identify the impact of RCN1 on the outcomes of patients with Glioblastoma multiforme (GBM). The study applied two public databases to require RNA sequencing data of Glioblastoma multiform samples with clinical data for the construction of a training set and a validation set, respectively. We used bioinformatic analyses to determine that RCN1 could be an independent factor for the overall survival of Glioblastoma multiform patients. In the training set, the study constructed a predictive prognostic model based on the combination of RCN1 with various clinical parameters for overall survival at 0.5-, 1.0-, and 1.5-years, as well as developed a nomogram, which was further validated by validation set. Pathways analyses indicated that RCN1 was involved in KEAS and MYC pathways and apoptosis. In vitro experiments indicated that RCN1 promoted cell invasion of Glioblastoma multiform cells. These results illustrated the prognostic role of RCN1 for overall survival in Glioblastoma multiform patients, indicated the promotion of RCN1 in cell invasion, and suggested the probability of RCN1 as a potential targeted molecule for treatment in Glioblastoma multiform.

RICE CENTRORADIALIS 1, a TFL1-like Gene, Responses to Drought Stress and Regulates Rice Flowering Transition.

BACKGROUND: The initiation of flowering transition in rice (Oryza sativa) is a complex process regulated by genes and environment. In particular, drought can interfere with flowering; therefore, many plants hasten this process to shorten their life cycle under water scarcity, and this is known as drought-escape response. However, rice has other strategies; for example, drought stress can delay flowering instead of accelerating it. RICE CENTRORADIALIS 1 (RCN1) is a TERMINAL FLOWER-like gene that influences rice flowering transition and spike differentiation. It interacts with 14-3-3 proteins and transcription factor OsFD1 to form a florigen repression complex that suppresses flowering transition in rice. RESULTS: In this study, we explored the role of RCN1 in the molecular pathway of drought-regulated flowering transition. The rcn1 mutant plants displayed early heading under both normal water and drought stress conditions, and they were more insensitive to drought stress than the wild-type plants. Abscisic acid (ABA) signaling-mediated drought-induced RCN1 is involved in this process. CONCLUSIONS: Thus, RCN1 plays an important role in the process of drought stress inhibiting flowering transition. It may worked by suppressing the protein function rather than transcription of HEADING DATE 3a.

Salivary NUS1 and RCN1 Levels as Biomarkers for Oral Squamous Cell Carcinoma Diagnosis.

BACKGROUND/AIM: Oral cancer may become advanced because of delay in diagnosis. In order to promote oral cancer screening, simple and highly reliable screening methods that can be implemented at general dental clinics are required. Herein we investigated differential salivary gene expression between oral squamous cell carcinoma (OSCC) patients and healthy volunteers (HV) to identify new biomarkers for OSCC detection. MATERIALS AND METHODS: Candidate genes were selected by microarrays, nuclear undecaprenyl pyrophosphate synthase 1 (NUS1) and reticulocalbin 1 (RCN1) were selected for further investigation. We used real-time quantitative reverse transcription PCR (qRT-PCR) to determine NUS1 and RCN1 expression levels in saliva and tissues. RESULTS: qRT-PCR analysis of clinical samples revealed that OSCC patients had significantly higher expression of salivary NUS1 and RCN1 than HV. CONCLUSION: A combination of NUS1 and RCN1 accurately distinguished patients from controls, and this combination can be implemented as a screening test for OSCC.

Overexpression of RCN1 correlates with poor prognosis and progression in non-small cell lung cancer.

We investigated the expression of reticulocalbin-1 (RCN1) and its prognostic significance in non-small cell lung cancer (NSCLC). RCN1 expression was evaluated by immunohistochemical analysis with tissue microarrays in NSCLC tissues and matched adjacent noncancerous tissues. Furthermore, quantitative polymerase chain reaction and Western blot were also used to examine the expression of RCN1. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, clone formation, and transwell assays were used to measure cell proliferation, migration, and invasion. Lastly, we used the Kaplan-Meier method and log-rank test to compare overall survival rates between the RCN1-high expression group and the RCN1-low expression group. In this study, immunohistochemistry by tissue microarray at RCN1 expression was significantly up-regulated in NSCLC tissues compared with adjacent noncancerous tissues. We further confirmed the up-regulation of RCN1 by quantitative polymerase chain reaction and Western blot assay. RCN1 expression level was closely related to lymph node metastasis (P < .001) and TNM stage (P = .012). Kaplan-Meier analysis showed that high RCN1 expression was remarkably associated with poor prognosis with NSCLC patients. A suppression of cell proliferation, migration, and invasion was obtained in A549 cells treated with RCN1 small interfering RNA. Our data indicate that RCN1 expression may have an vital role at promoting the occurrence of NSCLC, and it may be a vital molecular marker in the diagnosis and prognosis of NSCLC patients.

B-RAF and its novel negative regulator reticulocalbin 1 (RCN1) modulates cardiomyocyte hypertrophy.

AIM: Activation of the kinase RAF and its downstream targets leads to cardiomyocyte hypertrophy. It has been hypothesized that B-RAF might be the main activator of MEK in various cell types. Therefore, the aim of this study was to investigate the role of B-RAF and its modulating factors in cardiomyocyte hypertrophy. METHODS AND RESULTS: Neonatal rat cardiomyocytes were pre-treated with and without the specific B-RAF inhibitor SB590885 and then stimulated with phenylephrine to induce hypertrophy. Inhibition of B-RAF completely impeded the hypertrophic response and led to a significant reduction of MEK1/2 phosphorylation. By applying a eukaryotic cDNA expression screen, based on a dual-luciferase reporter assay for B-RAF activity measurement, we identified RCN1 as a new negative modulator of B-RAF activity. Adenovirus-mediated overexpression of reticulocalbin 1 (RCN1) completely impeded phenylephrine-induced hypertrophy and led to significantly reduced MEK1/2 phosphorylation. Conversely, adenoviral knockdown of RCN1 with a specific synthetic miRNA induced cardiomyocyte hypertrophy and significantly increased MEK1/2 phosphorylation. CONCLUSIONS: In summary, our results show that the inhibition of B-RAF abolishes cardiomyocyte hypertrophy and we identified RCN1 as novel negative modulator of cardiomyocyte hypertrophy by inhibition of the mitogen-activated protein kinase signalling cascade. Our results show that B-RAF kinase activity is essential for cardiac hypertrophy and RCN1, its newly identified negative regulator, abolishes hypertrophic response of cardiomyocytes in vitro.

Proteomic identification of Reticulocalbin 1 as potential tumor marker in renal cell carcinoma.

Renal cell carcinoma (RCC) biomarkers are necessary for diagnosis and prognosis. They serve to monitor therapy response and follow-up, as drug targets, and therapy predictors in personalized treatments. Proteomics is a suitable method for biomarker discovery. Here we investigate differential protein expression in RCC, and we evaluate Reticulocalbin 1 (RCN1) use as a new potential marker. Neoplastic and healthy tissue samples were collected from 24 RCC patients during radical nephrectomy. Seven specimens were firstly processed by proteomic analysis (2-DE and MALDI-TOF) and 18 differentially expressed proteins from neoplastic and healthy renal tissues were identified. Among them, RCN1 was over-expressed in all cancer specimens analyzed by proteomics. Consequently RCN1 use as a potential marker was further evaluated in all 24 donors. RCN1 expression was verified by Western blotting (WB) and immunohistochemistry (IHC). WB analysis confirmed RCN1 over-expression in 21 out of 24 tumor specimens, whereas IHC displayed focal or diffuse expression of RCN1 in all 24 RCC tissues. Thus RCN1 appears as a potential marker for clinical approaches. A larger histopathological trial will clarify the prognostic value of RCN1 in RCC. BIOLOGICAL SIGNIFICANCE: The present work aimed at finding new biomarkers for RCC - a life-threatening disease characterized by high incidence in Western countries - by performing differential proteomic analysis of neoplastic and normal renal tissues obtained from a small cohort of RCC patients. Some of the identified proteins have been previously associated to renal cancer however data confirming the possible use of these proteins in clinical practice are not available to date. By IHC we demonstrated that RCN1 could be easily employed in clinical practice, confirming RCN1 over-expression in RCC tissues of all examined patients, and weak protein expression in healthy renal tissues only in correspondence to the renal tubule section. These data indicate a promising role of RCN1 as a possible marker in RCC and indicate the proximal convoluted renal tubule as a putative origin point for RCC. Since IHC staining displayed different grades of intensity in tested tissues, we hypothesized that RCN1 could also be employed as a prognostic marker or as a response predictor for RCC-targeted therapy. To test such a hypothesis, a larger retrospective trial on paraffin-embedded tissues obtained from radical or partial nephrectomy of RCC patients is planned to be performed by our group.

Convergent Evolution of Calcineurin Pathway Roles in Thermotolerance and Virulence in Candida glabrata.

Candida glabrata is an emerging human fungal pathogen that is frequently drug tolerant, resulting in difficulties in treatment and a higher mortality in immunocompromised patients. The calcium-activated protein phosphatase calcineurin plays critical roles in controlling drug tolerance, hyphal growth, and virulence in diverse fungal pathogens via distinct mechanisms involving survival in serum or growth at host temperature (37° and higher). Here, we comprehensively studied the calcineurin signaling cascade in C. glabrata and found novel and uncharacterized functions of calcineurin and its downstream target Crz1 in governing thermotolerance, intracellular architecture, and pathogenesis in murine ocular, urinary tract, and systemic infections. This represents a second independent origin of a role for calcineurin in thermotolerant growth of a major human fungal pathogen, distinct from that which arose independently in Cryptococcus neoformans. Calcineurin also promotes survival of C. glabrata in serum via mechanisms distinct from C. albicans and thereby enables establishment of tissue colonization in a murine systemic infection model. To understand calcineurin signaling in detail, we performed global transcript profiling analysis and identified calcineurin- and Crz1-dependent genes in C. glabrata involved in cell wall biosynthesis, heat shock responses, and calcineurin function. Regulators of calcineurin (RCN) are a novel family of calcineurin modifiers, and two members of this family were identified in C. glabrata: Rcn1 and Rcn2. Our studies demonstrate that Rcn2 expression is controlled by calcineurin and Crz1 to function as a feedback inhibitor of calcineurin in a circuit required for calcium tolerance in C. glabrata. In contrast, the calcineurin regulator Rcn1 activates calcineurin signaling. Interestingly, neither Rcn1 nor Rcn2 is required for virulence in a murine systemic infection model. Taken together, our findings show that calcineurin signaling plays critical roles in thermotolerance and virulence, and that Rcn1 and Rcn2 have opposing functions in controlling calcineurin signaling in C. glabrata.