Vacuolar H+-ATPase and BZR1 form a feedback loop to regulate the homeostasis of BR signaling in Arabidopsis.

Brassinosteroid (BR) is a vital plant hormone that regulates plant growth and development. BRASSINAZOLE RESISTANT 1 (BZR1) is a key transcription factor in BR signaling, and its nucleocytoplasmic localization is crucial for BR signaling. However, the mechanisms that regulate BZR1 nucleocytoplasmic distribution and thus the homeostasis of BR signaling remain largely unclear. The vacuole is the largest organelle in mature plant cells and plays a key role in maintenance of cellular pH, storage of intracellular substances, and transport of ions. In this study, we uncovered a novel mechanism of BR signaling homeostasis regulated by the vacuolar H+-ATPase (V-ATPase) and BZR1 feedback loop. Our results revealed that the vha-a2 vha-a3 mutant (vha2, lacking V-ATPase activity) exhibits enhanced BR signaling with increased total amount of BZR1, nuclear-localized BZR1, and the ratio of BZR1/phosphorylated BZR1 in the nucleus. Further biochemical assays revealed that VHA-a2 and VHA-a3 of V-ATPase interact with the BZR1 protein through a domain that is conserved across multiple species. VHA-a2 and VHA-a3 negatively regulate BR signaling by interacting with BZR1 and promoting its retention in the tonoplast. Interestingly, a series of molecular analyses demonstrated that nuclear-localized BZR1 could bind directly to specific motifs in the promoters of VHA-a2 and VHA-a3 to promote their expression. Taken together, these results suggest that V-ATPase and BZR1 may form a feedback regulatory loop to maintain the homeostasis of BR signaling in Arabidopsis, providing new insights into vacuole-mediated regulation of hormone signaling.

Titration-based normalization of antibody amount improves consistency of ChIP-seq experiments.

Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is frequently utilized in chromatin biology and epigenetics. The challenge in experimental variability by unpredictable nature of usable input amounts from samples and undefined antibody titer in ChIP reaction still remains to be addressed. Here, we introduce a simple and quick method to quantify chromatin inputs and demonstrate its utility for normalizing antibody amounts to the optimal titer in individual ChIP reactions. For a proof of concept, we utilized ChIP-seq validated antibodies against the key enhancer mark, acetylation of histone H3 on lysine 27 (H3K27ac), in the experiments. The results indicate that the titration-based normalization of antibody amounts improves assay outcomes including the consistency among samples both within and across experiments for a broad range of input amounts.

Glucocorticoids unmask silent non-coding genetic risk variants for common diseases.

Understanding the function of non-coding genomic sequence variants represents a challenge for biomedicine. Many diseases are products of gene-by-environment interactions with complex mechanisms. This study addresses these themes by mechanistic characterization of non-coding variants that influence gene expression only after drug or hormone exposure. Using glucocorticoid signaling as a model system, we integrated genomic, transcriptomic, and epigenomic approaches to unravel mechanisms by which variant function could be revealed by hormones or drugs. Specifically, we identified cis-regulatory elements and 3D interactions underlying ligand-dependent associations between variants and gene expression. One-quarter of the glucocorticoid-modulated variants that we identified had already been associated with clinical phenotypes. However, their affected genes were 'unmasked' only after glucocorticoid exposure and often with function relevant to the disease phenotypes. These diseases involved glucocorticoids as risk factors or therapeutic agents and included autoimmunity, metabolic and mood disorders, osteoporosis and cancer. For example, we identified a novel breast cancer risk gene, MAST4, with expression that was repressed by glucocorticoids in cells carrying the risk genotype, repression that correlated with MAST4 expression in breast cancer and treatment outcomes. These observations provide a mechanistic framework for understanding non-coding genetic variant-chemical environment interactions and their role in disease risk and drug response.

The combined effects of microplastics and the heavy metal cadmium on the marine periphytic ciliate Euplotes vannus.

Microplastics could be grazed by marine organisms and possibly transferred to higher trophic levels along the microbial loop. Due to their size and capacity to concentrate heavy metals that trigger joint toxic effects, microplastics (MPs) have already become a severe threat to marine organisms. The detrimental effects of MPs on large marine organisms have been studied, but the combined toxicity of MPs and cadmium (Cd) on protozoan ciliates remains unclear. In the present study, we selected different diameters and concentrations of polystyrene microspheres (PS-MPs) and Cd2+ as model MPs and heavy metals to evaluate their single and combined effects on the periphytic marine ciliate Euplotes vannus in relation to carbon biomass and oxidative stress. The MPs were indeed ingested by Euplotes vannus and significantly reduced the abundance and carbon biomass of ciliate populations. Combined exposure to MPs and Cd2+ not only increased the bioaccumulation of Cd2+ in ciliates but also exacerbated the decrease in ciliate biomass by increasing oxidative stress and membrane damage. In comparison, the effects of nano-sized plastics (0.22 µm) were more harmful than those of micro-sized plastics (1.07 µm, 2.14 µm and 5.00 µm). A smaller size represents a higher potential for penetrating biological members and a stronger adsorption capacity for cadmium. These results provide new insight into the combined toxicity of microplastics and heavy metals on ciliated protozoa and lay a foundation for higher trophic levels and ecosystems.

Six-way decomposition of causal effects: Unifying mediation and mechanistic interaction.

The sufficient component cause (SCC) model and counterfactual model are two common methods for causal inference, each with their own advantages: the SCC model allows the mechanistic interaction to be detailed, whereas the counterfactual model features a systemic framework for quantifying causal effects. Hence, integrating the SCC and counterfactual models may facilitate the conceptualization of causation. Based on the marginal SCC (mSCC) model, we propose a novel counterfactual mSCC framework that includes the steps of definition, identification, and estimation. We further propose a six-way effect decomposition for assessing mediation and the mechanistic interaction. The results demonstrate that when all variables are binary, the six-way decomposition is an extension of four-way decomposition and that without agonism, the six-way decomposition is reduced to four-way decomposition. To illustrate the utility of the proposed decomposition, we apply it to a Taiwanese cohort to examine the mechanism of hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) with liver inflammation measured by alanine aminotransferase (ALT) as a mediator. Among the HCV-induced HCC cases, 62.27% are not explained by either mediation or interaction in relation to ALT; 9.32% are purely mediated by ALT; 16.53% are caused by the synergistic effect of HCV and ALT; and 9.31% are due to the mediated synergistic effect of HCV and ALT. In summary, we introduce an SCC model framework based on counterfactual theory and detail the required identification assumptions and estimation procedures; we also propose a six-way effect decomposition to unify mediation and mechanistic interaction analyses.

RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

Directed migration is essential for cell motility in many processes, including development and cancer cell invasion. RSKs (p90 ribosomal S6 kinases) have emerged as central regulators of cell migration; however, the mechanisms mediating RSK-dependent motility remain incompletely understood. We have identified a unique signaling mechanism by which RSK2 promotes cell motility through leukemia-associated RhoGEF (LARG)-dependent Rho GTPase activation. RSK2 directly interacts with LARG and nucleotide-bound Rho isoforms, but not Rac1 or Cdc42. We further show that epidermal growth factor or FBS stimulation induces association of endogenous RSK2 with LARG and LARG with RhoA. In response to these stimuli, RSK2 phosphorylates LARG at Ser1288 and thereby activates RhoA. Phosphorylation of RSK2 at threonine 577 is essential for activation of LARG-RhoA. Moreover, RSK2-mediated motility signaling depends on RhoA and -B, but not RhoC. These results establish a unique RSK2-dependent LARG-RhoA signaling module as a central organizer of directed cell migration and invasion.

The expression of tachykinin receptors in the human lower esophageal sphincter.

Mammalian tachykinins are a family of neuropeptides which are potent modulators of smooth muscle function with a significant contractile effect on human smooth muscle preparations. Tachykinins act via three distinct G protein-coupled neurokinin (NK) receptors, NK1, NK2 and NK3, coded by the genes TACR1, TACR2 and TACR3 respectively. The purpose of this paper was to measure the mRNA and protein expression of these receptors and their isoforms in the clasp and sling fibers of the human lower esophageal sphincter complex and circular muscle from the adjacent distal esophagus and proximal stomach. We found differences in expression between the different receptors within these muscle types, but the rank order of the receptor expression did not differ between the different muscle types. The rank order of the mRNA expression was TACR2 (α isoform)>TACR2 (ß isoform)>TACR1 (short isoform)>TACR1 (long isoform)>TACR3. The rank order of the protein expression was NK2>NK1>NK3. This is the first report of the measurement of the transcript and protein expression of the tachykinin receptors and their isoforms in the muscles of the human lower esophageal sphincter complex. The results provide evidence that the tachykinin receptors could contribute to the regulation of the human lower esophageal sphincter, particularly the TACR2 α isoform which encodes the functional isoform of the tachykinin NK2 receptor was the most highly expressed of the tachykinin receptors in the muscles associated with the lower esophageal sphincter.

Effect of combined treatment with cyclophosphamidum and allicin on neuroblastoma-bearing mice.

OBJECTIVE: To evaluate the efficacy of allicin combined with cyclophosphamide on neuroblastoma (NB)-bearing mice and explore the immunological mechanism in it. METHODS: A total of 30 NB-bearing mice were equally randomized into model group, cyclophosphamide group and combined therapy group, 10 nudemice were set as normal saline (NS) group. Cyclophosphamide group and combined therapy group were weekly injected with 60 mg/kg cyclophosphamide for four weeks; besides, combined therapy group was given with allicin (10 mg/kg/d) by gastric perfusion for 4 weeks; model group and NS group were given with the same volume of NS. Serum VEGF content was detected by ELISA pre-treating (0 d) and on the 3rd d, 14th d and 28th d; on 29th d, all mice were sacrificed and the tumor, liver, spleen and thymic tissues were weighted. Tumors were made into paraffin section for detecting tumor cell apoptosis and proliferation by TUNEL and BrdU method, respectively. Survival curves were drawn by Kaplan-Meier method. RESULTS: After treatment, both treatment groups relieved on viscera indexes, VEGF level, T cell subsets distribution and tumor growth and each index of combined therapy group was better than cyclophosphamide group (P<0.05 or 0.01); only combined therapy group could significantly increase the lifetime of NB-bearing mice (µ (2)=5.667, P=0.017). CONCLUSIONS: Allicin can improve T cell subsets distribution and inhibit VEGF expression through its immunomodulatory activity, thereby improve the efficiency on NB in coordination with cyclophosphamide.

Analysis of the Rit subfamily GTPase-mediated signaling and neuronal differentiation and survival.

The Rit subfamily of GTPases is a founding branch within the Ras family of small G-proteins and preserves unique sequences in the G2 effector loop domain and the C-terminus. Rit proteins regulate a diversity of signal transduction pathways, some of which are similar to and others of which differ from the pathways that are regulated by other Ras family GTPases. Rit proteins have been demonstrated to be essential regulators in neuronal differentiation and survival. Here, we describe the materials and methods utilized to characterize cellular signaling for the Rit subfamily of G-proteins in neuronal differentiation and survival.

Rit subfamily small GTPases: regulators in neuronal differentiation and survival.

Ras family small GTPases serve as binary molecular switches to regulate a broad array of cellular signaling cascades, playing essential roles in a vast range of normal physiological processes, with dysregulation of numerous Ras-superfamily G-protein-dependent regulatory cascades underlying the development of human disease. However, the physiological function for many "orphan" Ras-related GTPases remain poorly characterized, including members of the Rit subfamily GTPases. Rit is the founding member of a novel branch of the Ras subfamily, sharing close homology with the neuronally expressed Rin and Drosophila Ric GTPases. Here, we highlight recent studies using transgenic and knockout animal models which have begun to elucidate the physiological roles for the Rit subfamily, including emerging roles in the regulation of neuronal morphology and cellular survival signaling, and discuss new genetic data implicating Rit and Rin signaling in disorders such as cancer, Parkinson's disease, autism, and schizophrenia.

Putting the Rit in cellular resistance: Rit, p38 MAPK and oxidative stress.

Cells mobilize diverse signaling pathways to protect against stress-mediated injury. Ras family GTPases play critical roles in this process, controlling the activation and integration of multiple regulatory cascades. p38 mitogen-activated protein kinase (MAPK) signaling serves as a critical fulcrum in this process, regulating networks that stimulate cellular apoptosis but also promote cell survival. However, this functional dichotomy is incompletely understood, particularly regulation of p38-dependent survival. Here, we discuss our recent evidence that the Rit GTPase associates with and is required for stress-mediated activation of a scaffolded p38-MK2-HSP27-Akt pro-survival signaling cascade. Drosophila lacking D-Ric, a Rit homologue, are susceptible to a variety of environmental stresses, while embryonic fibroblasts derived from Rit knockout mice display blunted stress-dependent signaling and decreased viability. Conversely, expression of constitutively active Rit triggers p38-Akt-dependent cell survival. Together, our studies establish Rit as the central regulator of an evolutionarily conserved, p38-dependent signaling cascade that functions as a critical survival mechanism in response to stress.

Rit-mediated stress resistance involves a p38-mitogen- and stress-activated protein kinase 1 (MSK1)-dependent cAMP response element-binding protein (CREB) activation cascade.

The cAMP response element (CRE)-binding protein (CREB) is a key regulatory factor of gene transcription, and plays an essential role in development of the central nervous system and for neuroprotection. Multiple signaling pathways have been shown to contribute to the regulation of CREB-dependent transcription, including both ERK and p38 mitogen-activated protein (MAP) kinases cascades. Recent studies have identified the Ras-related small G-protein, Rit, as a central regulator of a p38-MK2-HSP27 signaling cascade that functions as a critical survival mechanism for cells adapting to stress. Here, we examine the contribution of Rit-p38 signaling to the control of stress-dependent gene transcription. Using a pheochromocytoma cell model, we find that a novel Rit-p38-MSK1/2 pathway plays a critical role in stress-mediated CREB activation. RNAi-mediated Rit silencing, or inhibition of p38 or MSK1/2 kinases, was found to disrupt stress-mediated CREB-dependent transcription, resulting in increased cell death. Furthermore, ectopic expression of active Rit stimulates CREB-Ser133 phosphorylation, induces expression of the anti-apoptotic Bcl-2 and Bcl(XL) proteins, and promotes cell survival. These data indicate that the Rit-p38-MSK1/2 signaling pathway may have an important role in the stress-dependent regulation of CREB-dependent gene expression.

Rem-GTPase regulates cardiac myocyte L-type calcium current.

RATIONALE: The L-type calcium channels (LTCC) are critical for maintaining Ca(2+)-homeostasis. In heterologous expression studies, the RGK-class of Ras-related G-proteins regulates LTCC function; however, the physiological relevance of RGK-LTCC interactions is untested. OBJECTIVE: In this report we test the hypothesis that the RGK protein, Rem, modulates native Ca(2+) current (I(Ca,L)) via LTCC in murine cardiomyocytes. METHODS AND RESULTS: Rem knockout mice (Rem(-/-)) were engineered, and I(Ca,L) and Ca(2+) -handling properties were assessed. Rem(-/-) ventricular cardiomyocytes displayed increased I(Ca,L) density. I(Ca,L) activation was shifted positive on the voltage axis, and ß-adrenergic stimulation normalized this shift compared with wild-type I(Ca,L). Current kinetics, steady-state inactivation, and facilitation was unaffected by Rem(-/-) . Cell shortening was not significantly different. Increased I(Ca,L) density in the absence of frank phenotypic differences motivated us to explore putative compensatory mechanisms. Despite the larger I(Ca,L) density, Rem(-/-) cardiomyocyte Ca(2+) twitch transient amplitude was significantly less than that compared with wild type. Computer simulations and immunoblot analysis suggests that relative dephosphorylation of Rem(-/-) LTCC can account for the paradoxical decrease of Ca(2+) transients. CONCLUSIONS: This is the first demonstration that loss of an RGK protein influences I(Ca,L) in vivo in cardiac myocytes.

Ras family small GTPase-mediated neuroprotective signaling in stroke.

Selective neuronal cell death is one of the major causes of neuronal damage following stroke, and cerebral cells naturally mobilize diverse survival signaling pathways to protect against ischemia. Importantly, therapeutic strategies designed to improve endogenous anti-apoptotic signaling appear to hold great promise in stroke treatment. While a variety of complex mechanisms have been implicated in the pathogenesis of stroke, the overall mechanisms governing the balance between cell survival and death are not well-defined. Ras family small GTPases are activated following ischemic insults, and in turn, serve as intrinsic switches to regulate neuronal survival and regeneration. Their ability to integrate diverse intracellular signal transduction pathways makes them critical regulators and potential therapeutic targets for neuronal recovery after stroke. This article highlights the contribution of Ras family GTPases to neuroprotective signaling cascades, including mitogen-activated protein kinase (MAPK) family protein kinase- and AKT/PKB-dependent signaling pathways as well as the regulation of cAMP response element binding (CREB), Forkhead box O (FoxO) and hypoxiainducible factor 1(HIF1) transcription factors, in stroke.

A rit GTPase-p38 mitogen-activated protein kinase survival pathway confers resistance to cellular stress.

Cells mobilize diverse signaling cascades to protect against stress-mediated injury. Ras family GTPases play a pivotal role in cell fate determination, serving as molecular switches to control the integration of multiple signaling pathways. p38 mitogen-activated protein kinase (MAPK) signaling serves as a critical fulcrum in this process, regulating networks that stimulate cellular apoptosis but also have the capacity to promote cell survival. However, relatively little is known concerning this functional dichotomy, particularly the regulation of p38-dependent survival pathways. Here, we demonstrate that the Rit GTPase promotes cell survival by directing an unexpected p38 MAPK-dependent AKT survival pathway. Following stress exposure, Rit small hairpin RNA interference (shRNAi)-treated cells display increased apoptosis and selective disruption of p38 MAPK signaling, while expression of constitutively activated Rit promotes p38-AKT-dependent cell survival. Rit, but not Ras or Rap GTPases, can associate with, and is critical for, stress-mediated activation of the scaffolded p38-MK2-HSP27-AKT prosurvival signaling complex. Together, our studies establish Rit as a central regulator of a p38 MAPK-dependent signaling cascade that functions as a critical cellular survival mechanism in response to stress.

Src-dependent TrkA transactivation is required for pituitary adenylate cyclase-activating polypeptide 38-mediated Rit activation and neuronal differentiation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent neuropeptide that possesses both neurotrophic and neurodevelopmental effects. Recently, the Rit GTPase was found to be activated by a novel Galpha/cAMP/exchange protein activated by cyclic AMP (Epac)-dependent signaling pathway and required for PACAP-dependent cAMP response element-binding protein activation and neuronal differentiation. However, Epac did not function as a Rit guanine nucleotide exchange factor (GEF), and the nature of the PACAP regulatory cascade remained unclear. Here, we show that PACAP-mediated Rit activation involves Src family kinase-dependent TrkA receptor transactivation. PACAP receptor (PACR1) stimulation triggered both G(i)alpha and G(s)alpha/cAMP/Epac regulatory cascades resulting in Src kinase activity, which in turn induced TrkA kinase tyrosine phosphorylation. Importantly, Src inhibition, or the lack of functional Trk receptors, was found to inhibit PACAP-mediated Rit activation, whereas constitutively active Src alone was sufficient to stimulate Rit-guanosine triphosphate levels. A single tyrosine (Y(499)) phosphorylation event was identified as critical to both PACAP-mediated transactivation and TrkA-dependent Rit activation. Accordingly, PACAP stimulation resulted in TrkA-dependent phosphorylation of both the Shc adaptor and son of sevenless (SOS)1/2 GEFs, and Rit activation was inhibited by RNA interference silencing of SOS1/2, implicating a TrkA/Shc/SOS signaling complex in Rit regulation. Together, these observations expand upon the nature of PACR1-mediated transactivation and identify TrkA-Rit signaling as a key contributor to PACAP-dependent neuronal differentiation.

Depolarization increases phosphatidylinositol (PI) 4,5-bisphosphate level and KCNQ currents through PI 4-kinase mechanisms.

A growing body of evidence shows that membrane phosphatidylinositol 4,5-bisphosphates (PtdIns(4,5)P(2), PIP(2)) play an important role in cell signaling. The presence of PIP(2) is fundamentally important for maintaining the functions of a large number of ion channels and transporters, and for other cell processes such as vesicle trafficking, mobility, and endo- and exocytosis. PIP(2) levels in the membrane are dynamically modulated, which is an important signaling mechanism for modulation of PIP(2)-dependent cellular processes. In this study, we describe a novel mechanism of membrane PIP(2) modulation. Membrane depolarization induces an elevation in membrane PIP(2), and subsequently increases functions of PIP(2)-sensitive KCNQ potassium channels expressed in Xenopus oocytes. Further evidence suggests that the depolarization-induced elevation of membrane PIP(2) occurs through increased activity of PI4 kinase. With increased recognition of the importance of PIP(2) in cell function, the effect of membrane depolarization in PIP(2) metabolism is destined to have important physiological implications.

Specificity of Gbetagamma signaling depends on Galpha subunit coupling with G-protein-sensitive K(+) channels.

Many neurotransmitters activate G-protein-gated inwardly rectifying K(+) (Kir3) channels by stimulating G-protein-coupled receptors. However, in native systems, only receptors coupled to pertussis-toxin (PTX)-sensitive G proteins (Gi/Go) have been shown to be able to activate Kir3 channels through the betagamma subunits of G proteins (Gbetagamma), whereas activation of receptors coupled to PTX-insensitive G proteins such as Gq or Gs do not activate Kir3 channels. The question remains as to how signaling specificity is achieved and what are its key determinants. In this study, we have used the Xenopus oocyte expression system to investigate specific activation of Kir3 channels by heterotrimeric G proteins. We have demonstrated the activation of Kir3.4 channels by agonist stimulation of non-PTX-sensitive G proteins under conditions of Galpha subunit overexpression. We present evidence to suggest a key role for the coupling efficiency of Galpha subunits in determining the specificity of Gbetagamma signaling to the channel.

Rit signaling contributes to interferon-gamma-induced dendritic retraction via p38 mitogen-activated protein kinase activation.

The proinflammatory cytokine interferon-gamma (IFNgamma) alters neuronal connectivity via selective regressive effects on dendrites but the signaling pathways that mediate this effect are poorly understood. We recently demonstrated that signaling by Rit, a member of the Ras family of GTPases, modulates dendritic growth in primary cultures of sympathetic and hippocampal neurons. In this study, we investigated a role for Rit signaling in IFNgamma-induced dendritic retraction. Expression of a dominant negative Rit mutant inhibited IFNgamma-induced dendritic retraction in cultured embryonic rat sympathetic and hippocampal neurons. In pheochromacytoma cells and hippocampal neurons, IFNgamma caused rapid Rit activation as indicated by increased GTP binding to Rit. Silencing of Rit by RNA interference suppressed IFNgamma-elicited activation of p38 MAPK in pheochromacytoma cells, and pharmacological inhibition of p38 MAPK significantly attenuated the dendrite-inhibiting effects of IFNgamma in cultured sympathetic and hippocampal neurons without altering signal transducer and activator of transcription 1 activation. These observations identify Rit as a downstream target of IFNgamma and suggest that a novel IFNgamma-Rit-p38 signaling pathway contributes to dendritic retraction and may, therefore, represent a potential therapeutic target in diseases with a significant neuroinflammatory component.

Pituitary adenylate cyclase-activating polypeptide 38-mediated Rin activation requires Src and contributes to the regulation of HSP27 signaling during neuronal differentiation.

Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) is a potent neuropeptide that acts through G-protein-coupled receptors. While it is well established that PACAP mediates both neurotrophic and neurodevelopmental effects, the signaling cascades that underlie these diverse actions remain incompletely characterized. Here we show that the Ras-related Rin GTP-binding protein, a GTPase that is expressed predominantly in neurons, is regulated by PACAP38 signaling, and loss-of-function analysis demonstrates that Rin makes an essential contribution to PACAP38-mediated pheochromocytoma cell differentiation. Rin is activated following stimulation of both Gsalpha and Gialpha cascades but does not rely upon cyclic AMP (cAMP)-, Ca(2+)-, or Epac-dependent signaling pathways. Instead, Rin is activated in a Src kinase-dependent manner. Surprisingly, Rin knockdown significantly inhibits PACAP38-mediated neurite outgrowth, without affecting mitogen-activated protein kinase signaling cascades. Instead, Rin loss attenuates PACAP38-mediated HSP27 activation by disrupting a cAMP-protein kinase A cascade. RNA interference-mediated HSP27 silencing suppresses both PACAP38- and Rin-mediated neurite outgrowth, while expression of a constitutively active Rin mutant increases both HSP27 protein and phospho-HSP27 levels, supporting a role for Rin-HSP27 signaling in neuronal differentiation. Together, these observations identify an unsuspected role for Rin in neuronal PACAP signaling and establish a novel Galpha-Src-Rin-HSP27 signal transduction pathway as a critical element in PACAP38-mediated neuronal differentiation signaling.