Rodriguez, C. D. | bioRxiv Science

bioRxiv2023

Interplay of RAP2 GTPase and the cytoskeleton in Hippo pathway regulation

The Hippo signaling is instrumental in regulating organ size, regeneration, and carcinogenesis. The cytoskeleton emerges as a primary Hippo signaling modulator. Its structural alterations in response to environmental and intrinsic stimuli control Hippo kinase cascade activity. However, the precise mechanisms underlying the cytoskeleton regulation of Hippo signaling are not fully understood. RAP2 GTPase is known to mediate the mechanoresponses of Hippo signaling via activating the core Hippo kinases LATS1/2 through MAP4Ks and MST1/2. Here we show the pivotal role of the reciprocal regulation between RAP2 GTPase and the cytoskeleton in Hippo signaling. RAP2 deletion undermines the responses of the Hippo pathway to external cues tied to RhoA GTPase inhibition and actin cytoskeleton remodeling, such as energy stress and serum deprivtion. Notably, RhoA inhibitors and actin disruptors fail to activate LATS1/2 effectively in RAP2-deficient cells. RNA sequencing highlighted differential regulation of both actin and microtubule networks by RAP2 gene deletion. Consistently, Taxol, a microtubule-stabilizing agent, was less effective in activating LATS1/2 and inhibiting cell growth in RAP2 and MAP4K4/6/7 knockout cells. In summary, our findings position RAP2 as a central integrator of cytoskeletal signals for Hippo signaling, which offers new avenues for understanding Hippo regulation and therapeutic interventions in Hippo-impaired cancers.

cell biology↗

bioRxiv2023

Hippo Signaling Modulates Mechano-inflammatory Responses of Chondrocytes

Knee osteoarthritis (KOA) is a degenerative disease resulting from mechanical overload, where direct physical impacts on chondrocytes play a crucial role in disease development by inducing inflammation and extracellular matrix degradation. However, the signaling cascades that sense these physical impacts and induce the pathogenic transcriptional programs of KOA remain to be defined, which hinders the identification of novel therapeutic approaches. Recent studies have implicated a crucial role of Hippo signaling in osteoarthritis. Since Hippo signaling senses mechanical cues, we aimed to determine its role in chondrocyte responses to mechanical overload. Here we show that mechanical loading induces the expression of inflammatory and matrix-degrading genes by activating the nuclear factor-kappaB (NF{kappa}B) pathway in a Hippo-dependent manner. Applying mechanical compressional force to 3-dimensional cultured chondrocytes activated NF{kappa}B and induced the expression of NF{kappa}B target genes for inflammation and matrix degradation (i.e., IL1{beta} and ADAMTS4). Interestingly, deleting the Hippo pathway effector YAP or activating YAP by deleting core Hippo kinases LATS1/2 blocked the NF{kappa}B pathway activation induced by mechanical loading. Consistently, treatment with a LATS1/2 kinase inhibitor abolished the upregulation of IL1{beta} and ADAMTS4 caused by mechanical loading. Mechanistically, mechanical loading activates Protein Kinase C (PKC), which activates NF{kappa}B p65 by phosphorylating its Serine 536. Furthermore, the mechano-activation of both PKC and NF{kappa}B p65 is blocked in LATS1/2 or YAP knockout cells, indicating that the Hippo pathway is required by this mechanoregulation. Additionally, the mechanical loading-induced phosphorylation of NF{kappa}B p65 at Ser536 is blocked by the LATS1/2 inhibitor Lats-In-1 or the PKC inhibitor AEB-071. Our study suggests that the interplay of the Hippo signaling and PKC controls NF{kappa}B-mediated inflammation and matrix degradation in response to mechanical loading. Chemical inhibitors targeting Hippo signaling or PKC can prevent the mechanoresponses of chondrocytes associated with inflammation and matrix degradation, providing a novel therapeutic strategy for KOA.

cell biology↗