Ca2+ signaling and the Hippo pathway: Intersections in cellular regulation

Abstract

The Hippo signaling pathway is a master regulator of organ size and tissue homeostasis. Hippo integrates a broad range of cellular signals to regulate numerous processes, such as cell proliferation, differentiation, migration and mechanosensation. Ca²⁺ is a fundamental second messenger that modulates signaling cascades involved in diverse cellular functions, some of which are also regulated by the Hippo pathway. Studies published over the last five years indicate that Ca²⁺ can influence core Hippo pathway components. Nevertheless, comprehensive understanding of the crosstalk between Ca²⁺ signaling and the Hippo pathway, and possible mechanisms through which Ca²⁺ regulates Hippo, remain to be elucidated. In this review, we summarize the multiple intersections between Ca²⁺ and the Hippo pathway and address the biological consequences.

Keywords: Calcium; Calmodulin; Hippo; S100; Signaling; YAP.

Figure 1.. Core components of the Hippo pathway.

A. Schematic diagram illustrating the interactions among the core components of the Hippo pathway. When Hippo is OFF, YAP and TAZ accumulate in the nucleus, where they bind to and activate TEAD to promote the expression of target genes. B. When Hippo is ON, SAV1 allows MST to phosphorylate LATS, and MOB1 binds to LATS to enhance its catalytic activity. LATS then phosphorylates YAP/TAZ, causing their sequestration in the cytoplasm, either through binding to adaptor proteins, such as 14–3-3, or by inducing YAP/TAZ degradation in the proteasome. In the absence of nuclear YAP/TAZ, TEAD, which is bound to the transcription cofactor VGL4, suppresses the expression of YAP/TAZ target genes. Green arrows represent activation. Figure generated in BioRender.

Figure 2.. Ca²⁺ homeostasis in cells.

Intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) are tightly regulated by a network of proteins and channels, such as Ca²⁺-binding proteins, Ca²⁺pumps (e.g, Ca²⁺-ATPase), Ca²⁺ channels (e.g., Piezo1) and exchangers (e.g., Na⁺/Ca²⁺ exchanger). Diverse stimuli, including membrane depolarization, mechanical forces, receptor/ligand interactions, and intracellular messengers, induce “ON” mechanisms. These reactions increase [Ca²⁺]_i by promoting the entry of Ca²⁺ from the extracellular environment or by the release of Ca²⁺ from intracellular stores, e.g., the ER, Golgi and mitochondria. The mobilized Ca²⁺ acts as a messenger to modulate numerous Ca²⁺-sensitive processes. When the ER Ca²⁺ reservoir is depleted (Low Ca²⁺), Ca²⁺ influx is triggered by store-operated Ca²⁺ entry (SOCE), which results from coupling between Orai channels and STIM. Once Ca²⁺ has carried out its signaling function, it is rapidly removed from the cytosol through “OFF” mechanisms, which either extrude Ca²⁺ to the extracellular space or sequester Ca²⁺ into internal stores. Blue arrows indicate “ON” mechanisms, red arrows depict “OFF” mechanisms and black arrows indicate induction. Ca²⁺ is depicted by blue circles. ER, endoplasmic reticulum; CaBP, Ca²⁺-binding proteins; TRP, transient receptor potential; PMCA, plasma membrane Ca²⁺ pumps; GPCR, G-protein coupled receptor; ROC, receptor-operated channel; VGCC, voltage-gated Ca²⁺ channel. The schematic depicts only the main transporters that regulate Ca²⁺signaling. The figure was generated in BioRender.

Figure 3.. Ca²⁺-binding proteins modulate Hippo activation.

A. Hippo inhibition by S100 proteins: (i) Activation of RAGE by extracellular S100A8/S100A9 activates FAK, which inhibits MST1. (ii) Binding of S100A1 to LATS1 inhibits its kinase activity towards YAP. (iii) In both cases, this increases active, non-phosphorylated YAP, which stimulates the expression of YAP/TEAD target genes. B. Hippo activation by S100 proteins. (i) S100A7 activates NF-κB signaling, which inhibits ΔNp63. (ii) This mechanism prevents ΔNp63-mediated reduction and stimulation of MST1 and YAP expression, respectively. (iii) Separately, S100A14 overexpression increases FAT1 cellular abundance. (iv) In turn, FAT1 stimulates Tao-mediated Hippo kinase activation. (v) Binding of Ca²⁺/S100B to Ndr1/2 stimulates their kinase activities, which may also increase YAP phosphorylation. C. Hippo activation by calmodulin (CaM). (i) Ca²⁺/CaM binds to both LATS1 and YAP in a ternary complex in which Ca²⁺/CaM directly stimulates LATS1 kinase activity. (ii) This increases phosphorylated YAP, which impairs its nuclear translocation. (iii) Ca²⁺/CaM also interacts with Ndr1/2 kinases, which could possibly stimulate Ndr1/2-catalyzed phosphorylation of YAP. D. YAP activation by CaM. (i) Activation of the Frizzled receptor initiates non-canonical Wnt signaling, which increases cytosolic Ca²⁺ concentration via Ca²⁺ release from intracellular stores. (ii) This increases Ca²⁺/CaM which, via activation of CaMKK and CaMKII, (iii) stimulates YAP nuclear activity. Green, red, and dotted arrows represent activation, inhibition, and decrease, respectively. ? indicates speculative mechanisms not experimentally demonstrated. Figure generated in BioRender.

Figure 4.. Ca²⁺ and Hippo signaling crosstalk.

A. Mechanical stimuli, such as shear stress, mechanical tension and substratum stiffness, elicit a cascade of signaling events by inducing an influx of Ca²⁺ through the activation of Ca²⁺-permeable mechanosensitive channels, including the TRPV family of proteins, Piezo1, and connexins. The increased [Ca²⁺]_i activates downstream effectors, RhoA/ROCK and subsequent actin remodeling. Active RhoA inhibits LATS1/2 phosphorylation, leading to nuclear translocation and transcriptional activation of YAP. Increased [Ca²⁺]_i also activates CaMKII via CaM. CaMKII may promote nuclear translocation of YAP. B. Store-operated calcium entry (SOCE) triggers an increase in [Ca²⁺]_i, leading to the activation of PKC-βII and ubiquitination of Merlin. Active PKC-βII stimulates the Hippo kinase cascade, while ubiquitinated Merlin promotes LATS1 activity, all resulting in increased YAP phosphorylation and inhibition of its nuclear activity. Green arrows represent activation. Dashed line depicts speculative mechanism. CaM, calmodulin; CaMKII, Ca²⁺/calmodulin-dependent kinase II; ER, endoplasmic reticulum; ROCK, Rho kinase; and TRPV, Transient Receptor Potential Vanilloid. Figure generated in BioRender.

Figure 5.. Ca²⁺ and Hippo crosstalk in health and disease.

A. MST1 phosphorylates Cx43 and attenuates its ability to release inflammatory molecules, thus protecting endothelial cells from atherosclerosis. B. Cholesterol loading in hepatocytes suppresses degradation of TAZ in the proteasome by β-TrCP, an E3 ubiquitin ligase, via an IP3R-Ca²⁺-RhoA pathway, thereby promoting TAZ-mediated liver fibrosis. C. The interaction of bacterial cell wall components with toll-like receptors (TLRs) on macrophages initiates a cascade of events. Piezo1 couples with TLRs, mediating the influx of Ca²⁺, which activates MST1. This leads to actin remodeling and culminates in phagocytosis of the bacteria by the macrophages. D. Carcinoma cells secrete macromolecules, which increase the viscosity of the extracellular environment. This induces membrane tension and increases [Ca²⁺]_i via TRPV channels. Ca²⁺ activates RhoA and promotes actin remodeling, leading to cancer cell migration and metastasis. Green, red and dashed arrows represent activation, inhibition, and decrease, respectively. CaMKII, Ca²⁺/calmodulin-dependent kinase II; IP3R, inositol 1,4,5-triphosphate (IP₃) receptor; TLR, Toll-like receptor; and TRPV4, Transient Receptor Potential Vanilloid 4. Figure generated in BioRender.