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Activation of TRPV4 modulates TGFβ signalling in a time-dependent manor. TC28a2 cells with SBE-nLUCp reporter were used to monitor TGFβ signalling. (A) Cells were stimulated with 10 ng/mL TGFβ3 or medium control, incubated for 15 min then stimulated with 100 nM GSK101 (activator) or DMSO control (vehicle) and then incubated for a further 3 h 45 min before
Source publication
The growth factor TGFβ and the mechanosensitive calcium-permeable cation channel TRPV4 are both important for the development and maintenance of many tissues. Although TRPV4 and TGFβ both affect core cellular functions, how their signals are integrated is unknown. Here we show that pharmacological activation of TRPV4 significantly increased the can...
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... cells are immortalised and have a more stable pattern of gene expression in contrast to primary chondrocytes, which show age-related changes in proliferation and gene expression as they de-differentiate when cultured [34]. As evidence for their suitability, TC28a2 cells were tested for TRPV4 expression using Western blotting ( Figure S2) and immunofluorescence, showing all TC28a2 cells expressed some TRPV4 protein (Fig- ure 1A). To demonstrate TRPV4 activation TC28a2 cells were loaded with the fluorescent Fluo8 calcium indicator and stimulated with a selective TRPV4 activator GSK101 [35]. ...
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... can be activated by selective agonists such as GSK101 and when TGFβ reporter cells were tested with GSK101 following TGFβ3 stimulation, it was found to enhance TGFβ signalling, as shown by increased SBE-nLUCp activity (Figures 2A and S3A) and increased nuclear translocation of SMAD2 ( Figure S4A,B). This GSK101 effect was saturated at 100 nM ( Figure S3A) and this concentration was used for subsequent experiments. ...
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... GSK101 effect was saturated at 100 nM ( Figure S3A) and this concentration was used for subsequent experiments. GSK219 did not affect TGFβ3-induced SBE-nLUCp activity (Figures 2A and S3B), but it was able to block the GSK101 increase in TGFβ3-induced SBE-nLUCp activity (Fig- ures 2A and S3C). Based on these data, 500 nM GSK219 was used in subsequent experiments to ensure effective TRPV4 inhibition. ...
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... GSK101 effect was saturated at 100 nM ( Figure S3A) and this concentration was used for subsequent experiments. GSK219 did not affect TGFβ3-induced SBE-nLUCp activity (Figures 2A and S3B), but it was able to block the GSK101 increase in TGFβ3-induced SBE-nLUCp activity (Fig- ures 2A and S3C). Based on these data, 500 nM GSK219 was used in subsequent experiments to ensure effective TRPV4 inhibition. ...
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... on these data, 500 nM GSK219 was used in subsequent experiments to ensure effective TRPV4 inhibition. Using siRNA to TRPV4 prevented the GSK101 enhancement of TGFβ signalling, showing that the effect was indeed facilitated entirely through TRPV4 (Figures 2B and S3D). cells with SBE-nLUCp reporter were used to monitor TGFβ signalling. ...
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... signalling showed a small increase when TRPV4 was activated, even in the absence of added TGFβ3 (Figure 2A). This was likely due to low endogenous expression of TGFβ1 as it could be prevented by siRNAs to TRPV4 or TGFB1 ( Figures 2C and S3E). ...
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... signalling showed a small increase when TRPV4 was activated, even in the absence of added TGFβ3 (Figure 2A). This was likely due to low endogenous expression of TGFβ1 as it could be prevented by siRNAs to TRPV4 or TGFB1 ( Figures 2C and S3E). Importantly siRNA to TGFB1 did not prevent TRPV4-mediated enhancement of TGFβ signalling in the presence of exogenous TGFβ3 ( Figure 2B). ...
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... was likely due to low endogenous expression of TGFβ1 as it could be prevented by siRNAs to TRPV4 or TGFB1 ( Figures 2C and S3E). Importantly siRNA to TGFB1 did not prevent TRPV4-mediated enhancement of TGFβ signalling in the presence of exogenous TGFβ3 ( Figure 2B). Furthermore, TRPV4 activation also enhanced TGFβ signalling mediated by exogenous TGFβ1, showing that the effect of TRPV4 was unaffected by which TGFβ ligand was driving the TGFβ signalling ( Figure S4C-D). ...
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... it was unclear at what part of the TGFβ signalling cascade TRPV4 might be having its effect, we investigated the sequence and timing of responses. In our data reported above, when TRPV4 was activated 15 min after TGFβ3 stimulation, an enhancement of TGFβ signalling activity was observed (Figure 2A,B). To determine over what time scale TRPV4 activation affected TGFβ signalling, we activated TRPV4 at different times relative to TGFβ3 stimulation and then measured SBE-nLUCp activity 4 h post TGFβ3 stimulation. ...
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... determine over what time scale TRPV4 activation affected TGFβ signalling, we activated TRPV4 at different times relative to TGFβ3 stimulation and then measured SBE-nLUCp activity 4 h post TGFβ3 stimulation. Consistent with Figure 2A, activation of TRPV4 15 or 30 min after TGFβ3 stimulation enhanced TGFβ signalling ( Figure 2E). However, TRPV4 activation 15 or 30 min prior to TGFβ3 stimulation had the opposite effect and significantly reduced TGFβ signalling (Fig- ures 2E and S5). ...
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... determine over what time scale TRPV4 activation affected TGFβ signalling, we activated TRPV4 at different times relative to TGFβ3 stimulation and then measured SBE-nLUCp activity 4 h post TGFβ3 stimulation. Consistent with Figure 2A, activation of TRPV4 15 or 30 min after TGFβ3 stimulation enhanced TGFβ signalling ( Figure 2E). However, TRPV4 activation 15 or 30 min prior to TGFβ3 stimulation had the opposite effect and significantly reduced TGFβ signalling (Fig- ures 2E and S5). ...
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... with Figure 2A, activation of TRPV4 15 or 30 min after TGFβ3 stimulation enhanced TGFβ signalling ( Figure 2E). However, TRPV4 activation 15 or 30 min prior to TGFβ3 stimulation had the opposite effect and significantly reduced TGFβ signalling (Fig- ures 2E and S5). The addition of TRPV4 activator close to TGFβ3 (±2 min), had no significant effect ( Figure 2E). ...
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... TRPV4 activation 15 or 30 min prior to TGFβ3 stimulation had the opposite effect and significantly reduced TGFβ signalling (Fig- ures 2E and S5). The addition of TRPV4 activator close to TGFβ3 (±2 min), had no significant effect ( Figure 2E). These data demonstrate that the effect of TRPV4 is rapid, transient and dependent upon the stage of TGFβ signal transduction. ...
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... TRPV4 was activated after TGFβ3 stimulation, an increase in TGFβ signalling was consistently observed 4 h post stimulation (Figure 2). Consistent with the observed rapid nuclear SMAD2 translocation ( Figure S4), SBE activity showed enhancement as early as 1 h post TGFβ stimulation (45 min post TRPV4 activation), which then decreases with time ( Figure 2F), and this decrease was also observed for primary bovine articular chondrocytes and murine ATDC5 cells ( Figure S4). ...
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... TRPV4 was activated after TGFβ3 stimulation, an increase in TGFβ signalling was consistently observed 4 h post stimulation (Figure 2). Consistent with the observed rapid nuclear SMAD2 translocation ( Figure S4), SBE activity showed enhancement as early as 1 h post TGFβ stimulation (45 min post TRPV4 activation), which then decreases with time ( Figure 2F), and this decrease was also observed for primary bovine articular chondrocytes and murine ATDC5 cells ( Figure S4). Most likely, this was about the earliest timepoint at which TGFβ3 can activate a transcriptional response since no enhanced SBE response was seen at 30 min ( Figure 2F). ...
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... with the observed rapid nuclear SMAD2 translocation ( Figure S4), SBE activity showed enhancement as early as 1 h post TGFβ stimulation (45 min post TRPV4 activation), which then decreases with time ( Figure 2F), and this decrease was also observed for primary bovine articular chondrocytes and murine ATDC5 cells ( Figure S4). Most likely, this was about the earliest timepoint at which TGFβ3 can activate a transcriptional response since no enhanced SBE response was seen at 30 min ( Figure 2F). These data indicate that the mechanism by which TRPV4 activation enhances TGFβ signalling results in changes in gene expression output within 45 min, but it has no effect, or a negative effect if TRPV4 activation occurs before the TGFβ signalling cascade is active. ...
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... TRPV4-mediated enhancement of TGFβ signalling occurred within 1 h ( Figure 2F), we investigated the involvement of key regulatory transcription factors. The TRRUST transcription factor database was used to identify enrichment for transcription factors known to control the genes whose expression increased following TGFβ3 stimulation and TRPV4 activation. ...
Citations
... In our study, the downregulation of Ap-1 was related to FOSL1, suggesting that, as expected, Sr can play a key role in inhibiting osteoclast differentiation. In addition, three down-regulated transcripts were observed to be related to the TGF-β signaling pathway, which is another major pathway involved in the metabolism, differentiation, proliferation, and survival of chondrocytes [29]. SMAD1/5/9 can also play a significant role by regulating a series of biological processes in the TGF-β pathway with the changes in their phosphorylation levels [30]. ...
Strontium (Sr) is a trace element found mainly in bone, and it performs a dual action by promoting bone formation and inhibiting bone resorption. Sr has been used to evaluate the gastrointestinal calcium (Ca) absorption capacity of dairy cows due to the similar physicochemical properties of the two elements. However, the possible effects of Sr on dairy cows remain unclear. This study aimed to explore the potential regulatory mechanism of Sr in bovine chondrocytes by performing transcriptomic and proteomic analyses. A total of 111 genes (52 up-regulated and 59 down-regulated) were identified as significantly altered (1.2-fold change and p < 0.05) between control and Sr-treated groups. Moreover, LC-MS-based proteomic analysis detected 286 changed proteins (159 up-regulated and 127 down-regulated) between the control and Sr-treated groups (1.2-fold change and p < 0.05). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations of a combination analysis of the transcriptomic and proteomic data revealed that the genes were predominantly involved in chondrocyte proliferation and differentiation, fat metabolism, the inflammation process, and immune responses. Overall, our data reveal a potential regulatory mechanism of strontium in bovine chondrocytes, thus providing further insights into the functions and application of Sr in ruminants.
... In fact, TRPV4 and TGF-β signaling have recently been shown to interact, with effects specific to the order in which they occur (Nims et al., 2021;O'Conor et al., 2014;Woods et al., 2021). Consistent with previous finding with hiPSCs housing the I604M TRPV4 mutations (Saitta et al., 2014), the altered TRPV4 activity in our hiPSC-derived chondrocytes could be altering their response to the TGFβ3 and BMP4 treatments. ...
Mutations in the TRPV4 ion channel can lead to a range of skeletal dysplasias. However, the mechanisms by which TRPV4 mutations lead to distinct disease severity remain unknown. Here, we use CRISPR-Cas9-edited human induced pluripotent stem cells (hiPSCs) harboring either the mild V620I or lethal T89I mutations to elucidate the differential effects on channel function and chondrogenic differentiation. We found that hiPSC-derived chondrocytes with the V620I mutation exhibited increased basal currents through TRPV4. However, both mutations showed more rapid calcium signaling with a reduced overall magnitude in response to TRPV4 agonist GSK1016790A compared to wildtype. There were no differences in overall cartilaginous matrix production, but the V620I mutation resulted in reduced mechanical properties of cartilage matrix later in chondrogenesis. mRNA sequencing revealed that both mutations upregulated several anterior HOX genes and downregulated antioxidant genes CAT and GSTA1 throughout chondrogenesis. BMP4 treatment upregulated several essential hypertrophic genes in WT chondrocytes; however, this hypertrophic maturation response was inhibited in mutant chondrocytes. These results indicate that the TRPV4 mutations alter BMP signaling in chondrocytes and prevent proper chondrocyte hypertrophy, as a potential mechanism for dysfunctional skeletal development. Our findings provide potential therapeutic targets for developing treatments for TRPV4-mediated skeletal dysplasias.
... The transforming growth factor β (TGFβ) superfamily comprises more than forty members including TGFβ, activin, and bone morphogenetic protein (BMP) (Chen et al., 2012) all of which play pivotal roles in the metabolism, differentiation, proliferation, and survival of chondrocytes (van Caam et al., 2016;Woods et al., 2021). SMAD family member (SMAD)-dependent signaling is a classical pathway of the TGFβ family and it involves binding of TGFβ to its tetrameric receptor comprised of activin receptor-like kinase 5 (ALK5) and TGFβ type II kinase receptor dimers. ...
The present study evaluated the effects of strontium (Sr) on proliferation and differentiation of chondrocytes isolated from dairy cows, and whether Sr exerts its effects via transforming growth factor β (TGFβ) signaling. The chondrocytes were isolated from patellar cartilage from newborn Holstein bull calves (n = 3, 1 day old, 38.0 ± 2.8 kg, fasting) within 15 min after euthanasia, and treated with different concentrations of Sr (0, 0.1, 1, and 10 μg/ml, as SrCl 2 ·6H 2 O). After pretreatment with or without activin receptor-like kinase 5 (ALK5) inhibitor (10 μM SB-505124) for 4 h, chondrocytes were incubated with Sr for another 4 h. Overall effects of Sr were evaluated relative to NaCl as the control. In contrast, the 1 μg/ml Sr-treated group served as the control to determine effects of preincubating with SB-505124. Western blot and qRT-PCR were used for measuring expression of proliferation-, differentiation-, and TGFβ1-responsive factors. Data were analyzed using one-way ANOVA in GraphPad Prism 7.0. Incubation with all doses of Sr increased TGFβ1/ALK5-induced SMAD3 phosphorylation, and at 10 μg/ml it inhibited ALK1-induced SMAD1/5/9 phosphorylation. Expression of mRNA and protein of the proliferation-responsive factors type Ⅱ Collagen α1 (COL2A1) and aggrecan (ACAN) was induced by Sr at 1 μg/ml. In contrast, Sr at 10 μg/ml inhibited the expression of differentiation-responsive factors type Ⅹ Collagen α1 (COL10A1) and secreted phosphoprotein 1 (SPP1), and at 1 μg/ml it had the same effect on alkaline phosphatase (ALPL) mRNA and protein levels. Cells were stained with PI/RNase Staining buffer to assess cell cycle activity using flow-cytometry. Incubation with Sr at 1 and 10 μg/ml induced an increase in the number of cells in the S-phase, leading to an increase in the proliferation index. Incubation with SB-505124 inhibited phosphorylation of SMAD3. Abundance of ACAN and COL2A1 mRNA and protein was lower when cells were pre-incubated with SB-505124. Overall, data indicated that Sr promotes proliferation and inhibits differentiation of primary chondrocytes by directing TGFβ1 signaling towards SMAD3 phosphorylation rather than SMAD1/5/9 phosphorylation. Whether these effects occur in vivo remains to be determined and could impact future application of Sr as an experimental tool in livestock.
... Indeed, Gilchrist and colleagues [6] adopted intermittent treatments when exposing monolayers of hMSC to TRPV4 modulators. The specific mechanism of TRPV4 enhancement regulation of aggrecan is not completely understood, but recent evidence suggests TRPV4 activation drives expression of SOX9 via Ca 2þ /calmodulin signalling, which interacts with the TGF-β3 pathway through JUN and SP1 transcription factors [37], which in turn regulates ACAN expression. On the other hand, intracellular TRPV4-dependant Ca 2þ overload has been associated with activation of β-catenin downstream signalling through AKT activation [38], inhibiting proteoglycans [39] and cartilage ECM production in neocartilage. ...
Objective
To evaluate the effect of Transient Receptor Potential Vanilloid 4 (TRPV4) cation channel modulation on mesenchymal stromal cell (MSC)-derived neocartilage.
Methods
RT-PCR was performed to evaluate mRNA levels of chondrogenic, hypertrophic and candidate mechanoresponsive genes in equine neocartilage sheets exposed to pulses of the TRPV4 agonist (GSK101) at different concentrations (N = 10). Biochemical assays and mechanical tests (double indentation and unconfined compression) evaluated neocartilage properties (N = 5).
Results
GSK101 treatment (1 nM) increased ACAN levels after treatment for 1-h per day for 3 days. No increase was detected for hypertrophic markers RUNX2, MMP13, MMP1, ALP or COL10A1 at this concentration. This treatment regimen also increased sGAG content and enhanced compressive properties compared to untreated controls. GSK101 showed no effect on candidate mechanoresponsive genes at the time-point of analysis.
Conclusions
Chemical activation of TRPV4 signalling can be used as a strategy to enhance matrix synthesis and maturation of MSC-derived engineered neocartilage and augment its load-bearing capacity.
... Previous studies suggest that activation of TRPV4 drives expression of Sox9 via Ca 2+ /calmodulin signaling, 10 which is known to play a role in cartilage homeostasis and interact with the TGF-β pathway. 43,44 TGF-β and TRPV4 signaling pathways demonstrate synergistic transcriptomic profiles, including targets of SMAD3, JUN, and SP1, while also displaying Ca 2+ /calmodulin-dependence. 44 Together with our results, these findings depict TRPV4 as an active member in chondrogenic development, where it directly drives chondrogenic gene expression, including Sox9, by Ca 2+ /calmodulin-transduced interaction with the TGF-β pathway 10,14 It is also of interest to note that activation of TRPV4 by GSK101 appears to require the presence of extracellular matrix molecules, 22 particularly hyaluronic acid, 45 which may be associated with the increased matrix production observed in GFP+ cells. ...
... Previous studies suggest that activation of TRPV4 drives expression of Sox9 via Ca 2+ /calmodulin signaling, 10 which is known to play a role in cartilage homeostasis and interact with the TGF-β pathway. 43,44 TGF-β and TRPV4 signaling pathways demonstrate synergistic transcriptomic profiles, including targets of SMAD3, JUN, and SP1, while also displaying Ca 2+ /calmodulin-dependence. 44 Together with our results, these findings depict TRPV4 as an active member in chondrogenic development, where it directly drives chondrogenic gene expression, including Sox9, by Ca 2+ /calmodulin-transduced interaction with the TGF-β pathway 10,14 It is also of interest to note that activation of TRPV4 by GSK101 appears to require the presence of extracellular matrix molecules, 22 particularly hyaluronic acid, 45 which may be associated with the increased matrix production observed in GFP+ cells. ...
Transient receptor potential vanilloid 4 (TRPV4) is a polymodal calcium-permeable cation channel that is highly expressed in cartilage and is sensitive to a variety of extracellular stimuli. The expression of this channel has been associated with the process of chondrogenesis in adult stem cells as well as several cell lines. Here, we used a chondrogenic reporter (Col2a1-GFP) in murine induced pluripotent stem cells (iPSCs) to examine the hypothesis that TRPV4 serves as both a marker and a regulator of chondrogenesis. Over 21 days of chondrogenesis, iPSCs showed significant increases in Trpv4 expression along with the standard chondrogenic gene markers Sox9, Acan, and Col2a1, particularly in the green fluorescent protein positive (GFP+) chondroprogenitor subpopulation. Increased gene expression for Trpv4 was also reflected by the presence of TRPV4 protein and functional Ca2+ signaling. Daily activation of TRPV4 using the specific agonist GSK1016790A resulted in significant increases in cartilaginous matrix production. An improved understanding of the role of TRPV4 in chondrogenesis may provide new insights into the development of new therapeutic approaches for diseases of cartilage, such as osteoarthritis, or channelopathies and hereditary disorders that affect cartilage during development. Harnessing the role of TRPV4 in chondrogenesis may also provide a novel approach for accelerating stem cell differentiation in functional tissue engineering of cartilage replacements for joint repair.
3D‐printed cell‐laden hydrogels as tissue constructs show great promise in generating living tissues for medicine. Currently, the maturation of 3D‐printed constructs into living tissues remains challenge, since commonly used hydrogels struggle to provide an ideal microenvironment for the seeded cells. In this study, a cell‐adaptable nanocolloidal hydrogel is created for 3D printing of maturable tissue constructs. The nanocolloidal hydrogel is composed of interconnected nanoparticles, which is prepared by the self‐assembly and subsequent photocrosslinking of the gelatin methacryloyl solutions. Cells can get enough space to grow and migrate within the hydrogel through squeezing the flexible nanocolloidal networks. Meanwhile, the nanostructure can promote the seeded cells to proliferate and produce matrix proteins through mechanotransduction. Using digital light process‐based 3D printing technology, it can rapidly customize cartilage tissue constructs. After implantation, these tissue constructs efficiently matured into cartilage tissues for the articular cartilage defect repair and ear cartilage reconstruction in vivo. The 3D printing of maturable tissue constructs using the nanocolloidal hydrogel shows potential for future clinical applications.
Graphene Oxide (GO) has been shown to increase the expression of key cartilage genes and matrix components within 3D scaffolds. Understanding the mechanisms behind the chondroinductive ability of GO is...
Vertebral malformations (VMs) pose a significant global health problem, causing chronic pain and disability. Verte-bral defects occur as isolated conditions or within the spectrum of various congenital disorders, such as Klippel-Feil syndrome, congenital scoliosis, spondylocostal dysostosis, sacral agenesis, and neural tube defects. Although both genetic abnormalities and environmental factors can contribute to abnormal vertebral development, our knowledge on molecular mechanisms of numerous VMs is still limited. Furthermore, there is a lack of resource that consolidates the current knowledge in this field. In this pioneering review, we provide a comprehensive analysis of the latest research on the molecular basis of VMs and the association of the VMs-related causative genes with bone developmental signaling pathways. Our study identifies 118 genes linked to VMs, with 98 genes involved in biological pathways crucial for the formation of the vertebral column. Overall, the review summarizes the current knowledge on VM genetics, and provides new insights into potential involvement of biological pathways in VM pathogenesis. We also present an overview of available data regarding the role of epigenetic and environmental factors in VMs. We identify areas where knowledge is lacking, such as precise molecular mechanisms in which specific genes contribute to the development of VMs. Finally, we propose future research avenues that could address knowledge gaps.
The ADAMTS superfamily is composed of secreted metalloproteases and structurally related non-catalytic ADAMTS-like proteins. A subset of this superfamily, including ADAMTS6, ADAMTS10 and ADAMTSL2, are involved in elastic fiber assembly and bind to fibrillin and other matrix molecules that regulate the extracellular bioavailability of the potent growth factor TGFβ. Fibrillinopathies, that can also result from mutation of these ADAMTS/L proteins, have been linked to disrupted TGFβ homeostasis. ADAMTS6 and ADAMTS10 are homologous metalloproteases with poorly characterized substrates where ADAMTS10 is thought to process fibrillin-2 and ADAMTS6 latent TGFβ-binding protein (LTBP)-1. In order to understand the contribution of ADAMTS6, and these other members of the ADAMTS/L family, to TGFβ homeostasis, we have analyzed the effects of ADAMTS6, ADAMTS10 and ADAMTSL2 expression on TGFβ activation. We found that their expression increases TGFβ activation in a dose dependent manner, following stimulation with mature TGFβ1. For ADAMTS6, the catalytically active protease is required for effective TGFβ activation, where ADAMTS6 cleaves LTBP3 as well as LTBP1, and binds to the large latent TGFβ complexes of LTBP1 and LTBP3. Furthermore, ADAMTS6 expression increases the mechanotension of cells which results in inactivation of the Hippo Pathway, resulting in an increased translocation of YAP/TAZ complex to the nucleus. Together these findings suggest that when the balance of TGFβ is perturbed ADAMTS6 can influence TGFβ activation via two mechanisms. It directly cleaves the latent TGFβ complexes and also acts indirectly, along with ADAMTS10 and ADAMTSL2, by altering the mechanotension of cells. Together this increases activation of TGFβ from large latent complexes which may contribute to disease pathogenesis.
