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Inhibition of ERK 1/2 Kinases Prevents Tendon Matrix Breakdown Ulrich Blache1,2,3, Stefania L

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Inhibition of ERK 1/2 Kinases Prevents Tendon Matrix Breakdown Ulrich Blache1,2,3, Stefania L www.nature.com/scientificreports OPEN Inhibition of ERK 1/2 kinases prevents tendon matrix breakdown Ulrich Blache1,2,3, Stefania L. Wunderli1,2,3, Amro A. Hussien1,2, Tino Stauber1,2, Gabriel Flückiger1,2, Maja Bollhalder1,2, Barbara Niederöst1,2, Sandro F. Fucentese1 & Jess G. Snedeker1,2* Tendon extracellular matrix (ECM) mechanical unloading results in tissue degradation and breakdown, with niche-dependent cellular stress directing proteolytic degradation of tendon. Here, we show that the extracellular-signal regulated kinase (ERK) pathway is central in tendon degradation of load-deprived tissue explants. We show that ERK 1/2 are highly phosphorylated in mechanically unloaded tendon fascicles in a vascular niche-dependent manner. Pharmacological inhibition of ERK 1/2 abolishes the induction of ECM catabolic gene expression (MMPs) and fully prevents loss of mechanical properties. Moreover, ERK 1/2 inhibition in unloaded tendon fascicles suppresses features of pathological tissue remodeling such as collagen type 3 matrix switch and the induction of the pro-fbrotic cytokine interleukin 11. This work demonstrates ERK signaling as a central checkpoint to trigger tendon matrix degradation and remodeling using load-deprived tissue explants. Tendon is a musculoskeletal tissue that transmits muscle force to bone. To accomplish its biomechanical function, tendon tissues adopt a specialized extracellular matrix (ECM) structure1. Te load-bearing tendon compart- ment consists of highly aligned collagen-rich fascicles that are interspersed with tendon stromal cells. Tendon is a mechanosensitive tissue whereby physiological mechanical loading is vital for maintaining tendon archi- tecture and homeostasis2. Mechanical unloading of the tissue, for instance following tendon rupture or more localized micro trauma, leads to proteolytic breakdown of the tissue with severe deterioration of both structural and mechanical properties3–5. Tese mechanisms are implicated in failed healing response and longer-term tendinopathies, with associated individual pain and socioeconomic burden that aficts our increasingly aging society6,7. At present, the underlying molecular mechanisms of tendinopathies are unknown and present a major roadblock to achieving targeted and efective therapies8–10. In the search for mechanisms that are involved in tendon degradation, tendon explants ofer a well-suited tissue model11,12. Tendon explants retain features of native tissue including the ECM architecture/structure, allow for highly controlled experimental conditions, and enable direct functional (mechanical) testing. In this sense, tendon explants overcome important limitations of traditional 2D cell culture systems, engineered 3D tissues and in vivo animal models13. Ex vivo, tendon explants lose their mechanical properties under load-deprived conditions due to proteolytic matrix degradation involving diferent proteases, such as matrix metalloproteases (MMP)12,14–18. We have previously used murine tendon fascicles to show that vascular like niches drive a cellular stress response and proteolytic tendon degradation12. However, the molecular pathways that govern ECM deg- radation and the subsequent loss of tendon biomechanical function remain unknown. In this work, we identify and probe the extracellular-signal regulated kinases ERK 1/2 as a central checkpoint that regulates proteolytic tendon matrix breakdown. Results Standard ex vivo tissue culture of tendon explants is widely documented to provoke tissue deterioration. In con- trast to the nutrient hyper-availability that characterizes standard tissue culture conditions, we employed more physiological ex vivo culture conditions comprising standard culture medium but at 3% oxygen and 29 °C—con- ditions we have identifed to maintain tendon fascicle viability while preserving native biomechanical properties12 (Fig. 1A). Here, we extend these fndings by exploring the potential molecular mechanisms that underpin the niche-dependent tendon degeneration in this model system. We started by re-examining our published transcriptome dataset of tendon fascicles (E-MTAB-7832), in which we have identifed ~ 3000 genes to be diferentially expressed between standard and reduced niche condi- tions. First, we applied the Expression2Kinase (E2K) pipeline to computationally infer key regulatory kinases 1Department of Orthopedics, Balgrist University Hospital, University of Zurich, Zurich, Switzerland. 2Institute for Biomechanics, ETH Zurich, Zurich, Switzerland. 3These authors contributed equally: Ulrich Blache and Stefania L. Wunderli. *email: [email protected] Scientifc Reports | (2021) 11:6838 | https://doi.org/10.1038/s41598-021-85331-1 1 Vol.:(0123456789) www.nature.com/scientificreports/ A B FGFR2FGFR3 TRKC EphB2 FGFR1 TRKB FGFR4 TRKA KDR FLT1 FMS EphB1 ROR2 EphA5 MUSK ROR1 KIT RET EphB3 DDR2 MER EphA3 AXL FLT4 EphA4 DDR1 TYRO3 PDGFRa Standard culture Reduced culture FLT3 IRR PDGFRb EphA6 IGF1R ErbB2 YES INSR MET EGFR EphB4 RON MLK3 SRC ROS MLK1 ALK TIE2 LYN EphA7 TIE1 LTK ErbB4 HCK FYN RYK MLK4 EphA8 CCK4 MLK2 TNK1 TYK2 LCK FGR ACK JAK1JAK2 Temperature ErbB3 37°C 29°C EphA2 JAK3 BLK TKL ZAP70 ANKRD3SgK288 DLK PYK2SYK LZK TK EphA1 LMR1 FAK LMR2 ALK4 RAF1 ITK FRK ZAK TGFbR1 TEC EphB6 KSR1 BRAF SRM RIPK2 KSR2 TXK IRAK1 BTK BRK LMR3 IRAK3 ALK7 LIMK1 ARAF BMX EphA10 LIMK2 BMPR1B TESK1 ILK BMPR1A Oxygen CTK RIPK3 TESK2 TAK1 ALK1 21% 3% CSK HH498 ALK2 ABL2 ACTR2 ABL1 FES IRAK2 ACTR2B FER RIPK1 TGFbR2 JAK3_b MAP3K2 JAK2_b LRRK1 MISR2 MAP3K3 LRRK2 BMPR2 MAP3K5 TYK2_b SuRTK106 IRAK4 MAP3K19 STE ANPa JAK1_b MAP3K7 DMEM DMEM ANPb MOS KHS1 MST1 YSK1 MST2 HSER HPK1 KHS2 MST3 SgK496 Wnk1 Medium GCK + 10% FBS + 10% FBS MAP3K6 MST4 DYRK2 PBK Wnk3 DYRK3 CYGD MAP3K4 NRBP2 Wnk2 HGK DYRK1A DYRK4 NRBP1 + 200µM AA + 200µM AA DYRK1B CYGF MAP3K1 OSR1 MINK STLK3 TNIK MLKL Wnk4 NRK STLK5 PEK STLK6 MYO3A SgK307 SLK PKR MYO3B HIPK1 HIPK3 GCN2 SgK424 LOK SCYL3 TAO1 HIPK2 SCYL1 COT TAO2 Mechanical SCYL2 NIK TAO3 PRP4 PAK1 yes no CLK4 HIPK4 HRI PAK3 PAK4 CLK1 CLIK1 PAK2 CLK2 IRE1 MAP2K5 PAK5 degradation IRE2 CLIK1L CLK3 TBCK PAK6 MAP2K7 RNAseL MAP2K1 GCN2_b MAP2K2 MSSK1 TTK SgK071 SRPK2 KIS MAP2K3 MYT1 MAP2K4 MAP2K6 CMGC SRPK1 CK2a1 Wee1 SgK196 CK1d MAK CK2a2 CDC7 Wee1B TTBK1 GSK3B ICK PRPK CK1e Haspin GSK3A MOK TTBK2 CK1a CDKL3 CK1a2 PINK1 CDKL2 SgK269 SgK493 VRK3 CK1g2 CDKL1 CDKL5 Erk7 SgK396 SgK223 CK1g1 CDKL4 Erk4 Slob SgK110 NLK Erk3 CK1g3 SgK069 PIK3R4 BUB1 Erk5 SBK CK1 IKKa BUBR1 VRK1 Erk1 IKKb CDK7 IKKe PLK4 VRK2 Erk2 p38g TBK1 MPSK1 JNK1 PITSLRE TLK2 p38d JNK3JNK2 CDK10 GAK PLK3 AAK1 ULK3 TLK1 p38b CDK8 CaMKK1 CDK11 PLK1 PLK2 p38a CDK4 CCRK BARK1 BIKE CaMKK2 RHOK CDK6 ULK1 BARK2 Fused GPRK5 GPRK7 PFTAIRE2 ULK2 ULK4 SgK494 PFTAIRE1 CDK9 NEK6 GPRK6 GPRK4 NEK7 RSKL1 PCTAIRE2 NEK8 NEK10 SgK495 PASK PDK1 MSK1 RSKL2 RSK1 NEK9 LKB1 MSK2 PCTAIRE1PCTAIRE3 CDK5CRK7CHED RSK4 RSK2 DYRK2 NEK2 CHK1 p70S6K RSK3 AKT2 NEK11 AurA p70S6Kb AKT1 DYRK3 CDC2 AKT3 CDK3 NEK4 SGK1 DYRK1A CDK2 AurB DYRK4 Trb3 PIM1 PIM2 AurC PKN1 SGK2SGK3 NEK3 LATS1 PKG2 PKN2 DYRK1B Trb2 PIM3 LATS2 PKG1 NEK5 NDR1 PKN3 Trb1 Obscn_b NDR2 PRKY YANK1 PRKX NEK1 SPEG_b MAST3 PKCh Obscn MASTL PKCz PKCe STK33 YANK2 PKCi PKCd PEK SPEG YANK3 PKCt PKACg PKACa PKCg TTN MAST2 PKACb PKR Trio smMLCK HIPK3 Trad ROCK1 PKCa HIPK1 GCN2 TSSK4 CHK2 HUNK ROCK2 PKCb skMLCK SNRK DMPK1 HIPK2 SSTK MAST4 MAST1 SCY DRAK2 NIM1 CRIK TSSK3 PKD2 PRP4 DRAK1 DCAMKL3 AGC CLK4 HIPK4 HRI SgK085 TSSK1 DCAMKL1 DMPK2 TSSK2 PKD1 DCAMKL2 CLK1 CLIK1 caMLCK MELK PKD3 CASK IRE1 DAPK3 DAPK2 VACAMKL MRCKb CLK2 CLIK1L PHKg1 DAPK1 MAPKAPK5 PSKH1 MRCKa IRE2 MNK1 PHKg2 TBCK AMPKa2 PSKH2 CLK3 MNK2 MAPKAPK2 AMPKa1 CaMK2g CaMK2a CaMK2b RNAseL BRSK2 MAPKAPK3CaMK2d CaMK4 BRSK1 RSK4_b TTK NuaK2 MSSK1 SgK071 NuaK1 RSK1_b MSK1_b KIS MSK2_b CaMK1b SRPK2 QSK RSK2_b RSK3_b CMGC SRPK1 CaMK1g CK2a1 SIK MAK QIK CK2a2 CDC7 CAMK CaMK1a ICK CaMK1d GSK3B MARK4 GSK3A MOK MARK1 MARK3 CDKL3 MARK2 CDKL2 S CDKL1 CDKL5 Erk7 CDKL4 Erk4 NLK Erk3 Erk5 Erk1 CDK7 Erk2 p38g JNK1 PITSLRE p38d JNK3JNK2 CDK10 p38b CDK8 CDK11 p38a CDK4 CCRK CDK6 PFTAIRE2 -log10(p-value) PFTAIRE1 CDK9 PCTAIRE2 PCTAIRE1PCTAIRE3 CDK5CRK7CHED CDC2 CDK3 CDK2 Min Max CD 2.5 2.0 K 1.5 Tubulin 1.0 p-ERK 1/2 p-ERK / t-ER 0.5 t-ERK 1/2 0.0 e Fresh tissue Fresh tissue StandardReduced cultur culture StandardReduced culture culture Figure 1. Niche-dependent ERK 1/2 activity and phosphorylation in load-deprived tendon explant cultures. (A) Overview of experimental conditions and starting phenotypes. DMEM = Dulbecco’s modifed Eagle’s medium, FBS = fetal bovine serum, AA = ascorbic acid. (B) Upstream kinase enrichment prediction based on the transcriptome data set E-MTAB-7832 (ArrayExpress), which analyses diferentially expressed genes in tendon tissues cultured ex vivo in standard and reduced niches. Kinome tree dendrogram mapping of all the predicted protein kinases generated by Coral42. Te circle color and size refect the enrichment signifcance with red color encoding higher signifcance. (C) Bar plots show the 20 most signifcantly enriched kinases. Red bars represent the ERK 1 and 2 kinases. GraphPad Prism (version 8.4.3) was used to generate the fgure. (D) Western blot validation of ERK 1/2 phosphorylation (ERK 1: Tr202/Tyr204; ERK 2: Tr185/Tyr187) in freshly isolated or cultured tendon fascicles (12 days). Full Western blot images are provided in the supplementary material (Supplementary Fig. 3). Quantifcation of band pixel intensities of phosphorylated ERK (p-ERK 1/2) compared to the total ERK (t-ERK). GraphPad Prism (version 8.4.3) was used to perform statistical analyses and generate the fgure. Bars show mean values + SD, with individual replicate values as data points, N = 8. Repeated measure ANOVA with Tukey’s multiple comparisons test: **p < 0.01, ***p < 0.001. Scientifc Reports | (2021) 11:6838 | https://doi.org/10.1038/s41598-021-85331-1 2 Vol:.(1234567890) www.nature.com/scientificreports/ upstream of the diferentially expressed genes (DEG). E2K algorithm predicted ~ 100 upstream protein kinases that are likely responsible for regulating DEG. Mapping the identifed kinases into Kinome phylogenetic tree showed that the majority of these kinase hits clustered around the CMGC kinases, which is a large kinase group including the mitogen-activated protein kinase (MAPK) family (Fig.
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