TGF‐β enhances Sox9 protein expression as well as downstream targets that regulate extracellular matrix production in bovine articular chondrocytes (BACs). The extracellular matrix protein 3‐Prime‐Phoshoadenosine 5‐Prime‐Phosphosulfate Synthase 2 (Papss2) has been associated with spondyloepimetaphyseal dysplasias in humans and is required for proper sulfation of proteoglycans in cartilage. Our work and that of others has shown that TGF‐β can regulate Papss2 expression in articular cartilage. We aim to identify whether Sox9 is involved downstream of TGF‐β in the regulation of Papss2 mRNA expression. Using primary BACs in micromass cultures, we analyzed the effect of TGF‐β (5ng/ml) on chondrocyte phenotype, Sox9 protein expression, stability, cellular localization and activity. We also assessed the effect of TGF‐β and/or Sox9 overexpression on the mRNA expression of Papss2, Prg4 and GAPDH via quantitative real‐time PCR. We found that treatment with TGF‐β enhanced proteoglycan content and delayed chondrocyte hypertrophic differentiation in BACs. We also observed an increase in Sox9 protein levels and stability, without changes in mRNA. The increase in Sox9 protein correlated with enhanced Col2a‐reporter activity. Importantly, we found Papss2 to be upregulated via Sox9 when wild‐type (WT)‐Sox9 was overexpressed via adenoviral transduction (50 MOI/cell). Interestingly, treatment +/‐ TGF‐β when Sox9 was overexpressed did not further increase Papss2 mRNA, supporting the hypothesis that Sox9 works downstream of TGF‐ β. However, TGF‐β, but not Sox9, increased Prg4 mRNA expression, suggesting specificity of signaling. A GAPDH control was examined and remained unchanged in response to either treatment. The regulation of Papss2 by Sox9 represents a novel paradigm involving TGF‐β‐dependent signaling in articular chondrocytes. Grant Funding Source : Supported by NIAMS‐ NIH
Convergent extension (CE) is a fundamental morphogenetic process where oriented cell behaviors lead to polarized extension of diverse tissues. In vertebrates, regulation of CE requires both non-canonical Wnt, its co-receptor Ror, and "core members" of the planar cell polarity (PCP) pathway. PCP was originally identified as a mechanism to coordinate the cellular polarity in the plane of static epithelium, where core proteins Frizzled (Fz)/ Dishevelled (Dvl) and Van Gogh-like (Vangl)/ Prickel (Pk) partition to opposing cell cortex. But how core PCP proteins interact with each other to mediate non-canonical Wnt/ Ror signaling during CE is not clear. We found previously that during CE, Vangl cell-autonomously recruits Dvl to the plasma membrane but simultaneously keeps Dvl inactive. In this study, we show that non-canonical Wnt induces Dvl to transition from Vangl to Fz. PK inhibits the transition, and functionally synergize with Vangl to suppress Dvl during CE. Conversely, Ror is required for the transition, and functionally antagonizes Vangl. Biochemically, Vangl interacts directly with both Ror and Dvl. Ror and Dvl do not bind directly, but can be cofractionated with Vangl. We propose that Pk assists Vangl to function as an unconventional adaptor that brings Dvl and Ror into a complex to serves two functions: 1) simultaneously preventing both Dvl and Ror from ectopically activating non-canonical Wnt signaling; and 2) relaying Dvl to Fz for signaling activation upon non-canonical Wnt induced dimerization of Fz and Ror.
Convergent extension (CE) is a fundamental morphogenetic process where oriented cell behaviors lead to polarized extension of diverse tissues. In vertebrates, regulation of CE requires both non-canonical Wnt, its co-receptor Ror, and “core members” of the planar cell polarity (PCP) pathway. PCP was originally identified as a mechanism to coordinate the cellular polarity in the plane of static epithelium, where core proteins Frizzled (Fz)/ Dishevelled (Dvl) and Van Gogh-like (Vangl)/ Prickel (Pk) partition to opposing cell cortex. But how core PCP proteins interact with each other to mediate non-canonical Wnt/ Ror signaling during CE is not clear. We found previously that during CE, Vangl cell-autonomously recruits Dvl to the plasma membrane but simultaneously keeps Dvl inactive. In this study, we show that non-canonical Wnt induces Dvl to transition from Vangl to Fz. PK inhibits the transition, and functionally synergize with Vangl to suppress Dvl during CE. Conversely, Ror is required for the transition, and functionally antagonizes Vangl. Biochemically, Vangl interacts directly with both Ror and Dvl. Ror and Dvl do not bind directly, but can be cofractionated with Vangl. We propose that Pk assists Vangl to function as an unconventional adaptor that brings Dvl and Ror into a complex to serves two functions: 1) simultaneously preventing both Dvl and Ror from ectopically activating non-canonical Wnt signaling; and 2) relaying Dvl to Fz for signaling activation upon non-canonical Wnt induced dimerization of Fz and Ror.
Convergent extension (CE) is a fundamental morphogenetic process where oriented cell behaviors lead to polarized extension of diverse tissues. In vertebrates, regulation of CE requires both non-canonical Wnt, its co-receptor Ror, and “core members” of the planar cell polarity (PCP) pathway. PCP was originally identified as a mechanism to coordinate the cellular polarity in the plane of static epithelium, where core proteins Frizzled (Fz)/ Dishevelled (Dvl) and Van Gogh-like (Vangl)/ Prickel (Pk) partition to opposing cell cortex. But how core PCP proteins interact with each other to mediate non-canonical Wnt/ Ror signaling during CE is not clear. We found previously that during CE, Vangl cell-autonomously recruits Dvl to the plasma membrane but simultaneously keeps Dvl inactive. In this study, we show that non-canonical Wnt induces Dvl to transition from Vangl to Fz. PK inhibits the transition, and functionally synergize with Vangl to suppress Dvl during CE. Conversely, Ror is required for the transition, and functionally antagonizes Vangl. Biochemically, Vangl interacts directly with both Ror and Dvl. Ror and Dvl do not bind directly, but can be cofractionated with Vangl. We propose that Pk assists Vangl to function as an unconventional adaptor that brings Dvl and Ror into a complex to serves two functions: 1) simultaneously preventing both Dvl and Ror from ectopically activating non-canonical Wnt signaling; and 2) relaying Dvl to Fz for signaling activation upon non-canonical Wnt induced dimerization of Fz and Ror.
Convergent extension (CE) is a fundamental morphogenetic mechanism that underlies numerous processes in vertebrate development, and its disruption can lead to human congenital disorders such as neural tube closure defects. The dynamic, oriented cell intercalation during CE is regulated by a group of core proteins identified originally in flies to coordinate epithelial planar cell polarity (PCP). The existing model explains how core PCP proteins, including Van Gogh (Vang) and Dishevelled (Dvl), segregate into distinct complexes on opposing cell cortex to coordinate polarity among static epithelial cells. The action of core PCP proteins in the dynamic process of CE, however, remains an enigma. In this report, we show that Vangl2 (Vang-like 2) exerts dual positive and negative regulation on Dvl during CE in both the mouse and Xenopus. We find that Vangl2 binds to Dvl to cell-autonomously promote efficient Dvl plasma membrane recruitment, a pre-requisite for PCP activation. At the same time, Vangl2 inhibits Dvl from interacting with its downstream effector Daam1 (Dishevelled associated activator of morphogenesis 1), and functionally suppresses Dvl → Daam1 cascade during CE. Our finding uncovers Vangl2-Dvl interaction as a key bi-functional switch that underlies the central logic of PCP signaling during morphogenesis, and provides new insight into PCP-related disorders in humans.
Abstract Convergent extension (CE) is a fundamental morphogenetic process where oriented cell behaviors lead to polarized extension of diverse tissues. In vertebrates, regulation of CE requires both non-canonical Wnt, its co-receptor Ror, and “core members” of the planar cell polarity (PCP) pathway. PCP was originally identified as a mechanism to coordinate the cellular polarity in the plane of static epithelium, where core proteins Frizzled (Fz)/ Dishevelled (Dvl) and Van Gogh-like (Vangl)/ Prickel (Pk) partition to opposing cell cortex. But how core PCP proteins interact with each other to mediate non-canonical Wnt/ Ror signaling during CE is not clear. We found previously that during CE, Vangl cell-autonomously recruits Dvl to the plasma membrane but simultaneously keeps Dvl inactive. In this study, we show that non-canonical Wnt induces Dvl to transition from Vangl to Fz. PK inhibits the transition, and functionally synergize with Vangl to suppress Dvl during CE. Conversely, Ror is required for the transition, and functionally antagonizes Vangl. Biochemically, Vangl interacts directly with both Ror and Dvl. Ror and Dvl do not bind directly, but can be cofractionated with Vangl. We propose that Pk assists Vangl to function as an unconventional adaptor that brings Dvl and Ror into a complex to serves two functions: 1) simultaneously preventing both Dvl and Ror from ectopically activating non-canonical Wnt signaling; and 2) relaying Dvl to Fz for signaling activation upon non-canonical Wnt induced dimerization of Fz and Ror.
