Yap is required for the tendon healing response
Mor Grinstein1, Heather Dingwall2, Ronen Schweitzer3, Terence Capellini2, Jenna Galloway1,4
1.
Center for Regenerative Medicine Massachusetts General Hospital and Harvard Stem Cell Institute.2. Department of Human Evolutionary Biology, Harvard
University. 3. Portland Shriners Research Center, Oregon Health and Science University 4.Department of Orthopaedic Surgery, Massachusetts General
Hospital, Boston.
ABSTRACT INTRODUCTION: Tendons connect and transfer force between the muscles and bones of the body, making them highly prone to injury.
Tendon and ligament injuries encompass acute tears and rupture due to chronic degeneration and deterioration of the tissue. Although there are surgical
treatments for tendon injuries, they can have a high failure rate and result in chronic pain and other debilitating side effects. Currently, we have limited
knowledge about the molecular mechanisms regulating tendon healing. One particular pathway, the Hippo pathway, has received attention because of its role
in regulating tissue size, regeneration, and mechanotransduction. The Hippo pathway consists of the key transcriptional effector Yap, Yes associated Kinase,
which is an important node in interpreting extra- and inter-cellular cues such as cell-cell, growth factor or mechanical signals and coordinating changes in
transcriptional regulation, cell fate, and proliferation. The tendon has a rich extracellular environment with unique cell-cell contacts and has also been found
to be regulated by mechanical signals. However, there is little understanding of the key molecular pathways that regulate the tendon cells during
maintenance and healing, and no characterization of Yap function in tendon biology. We believe the Yap pathway has an important and specific function in
the adult tendon by integrating cues from the extracellular environment to maintain and coordinate cell behaviors and gene expression in the tissue during
homeostasis and repair. For these reasons, we sought to define the functional role of Yap in adult tendons Our hypothesis is that Yap is a major regulator of
tendon cell proliferation and differentiation during the healing process and we propose to test this using gain and loss of function studies in mice.
METHODS: For our injury model, we are using a 300-micron biopsy-punch of the adult Achilles tendon. We analyzed the tendon healing process by
immunohistochemistry and qRT-PCR at 10, 20 and 30 days after injury. Immediately following tendon injury, BrdU was administered continuously to label
all proliferating cells. To perform Yap loss and gain of function studies specifically in the Scx+ tendon cells, we generated Scx-CreERt2; Yap fl/fl and ScxCre;TetO-Yap;Rosa-rtTA mice, respectively. All animal work was approved by the MGH IACUC, which is accredited by AAALAC and meets the NIH
standards.
RESULTS SECTION: Following an acute Achilles tendon injury, we observed increased and co-localized expression of Sox9 and Yap in proliferative cells
responding to the injury. Consistent with our immunohistochemistry results, Yap and Sox9 transcripts were increased by qRT-PCR in injured compared with
uninjured tendons at 20 days post injury. In the Yap loss-of-function mice (ScxCreER; Yap fl/fl), we found a decrease in Sox9 expression and proliferating
cells at the injury site compared to control injured tendons. In contrast, the gain-of-function mice (Scx-Cre;TetO-Yap;Rosa-rtTA) displayed an increase in
Sox9 expression and proliferating cells even in the uninjured Achilles tendons. In addition, the injured tendons expressing activated Yap appeared to have an
abnormal matrix deposition at the injury site compared with injured controls.
DISCUSSION: These results suggest that the Yap signaling pathway is a key regulator of tendon repair and is specifically required in Scx+ cells for gene
expression changes and proliferation following injury. Further analysis of this pathway in tendon homeostasis and injury response will help to better define
the molecular mechanisms underlying tendon healing.
: SIGNIFICANCE Our research identifies the Hippo signaling pathway as a key new regulator of the tendon healing response. Further mechanistic analysis
of the role of YAP signaling in tendon healing will allow us to better understand tendon biology and repair mechanisms, ultimately impacting the
development of regenerative solutions to tendon injury.
ACKNOWLEDGEMENTS: This work is supported by: Human Frontiers Science Program (HFSP) and the American Federation for Aging Research
(AFAR).
1
Uninjured tendon A Injured tendon B Figure 1: A,B Immunohistochemistry of SOX9 and YAP in injured tendon and uninjured
contralateral tendons. C. RT-qPCR shows increased Sox9 and Yap in injured compared with
control tendons.
Figure 2: A,B Immunohistochemistry for SOX9 and BrdU in transverse injured tendon sections of
control (A, B), Yap loss of function (C,D), and Yap gain of function (E,F) Immunohistochemistry
in transverse injured tendon sections of SOX9 and BrdU in gain of function
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A C Wild Type C ScxCreER;YAPF/F E D B D ORS 2017 Annual Meeting Poster No.0636
F ScxCre;TetOYAP