Tonic ATP-mediated growth suppression in peripheral nerve glia requires arrestin-PP2 and is evaded in NF1

Abstract Normal Schwann cells (SCs) are quiescent in adult nerves, when ATP is released from the nerve in an activity dependent manner. We find that suppressing nerve activity in adult nerves causes SC to enter the cell cycle. In vitro, ATP activates the SC G-protein coupled receptor (GPCR) P2Y2. Downstream of P2Y2, β-arrestin-mediated signaling results in PP2-mediated de-phosphorylation of AKT, and PP2 activity is required for SC growth suppression. NF1 deficient SC show reduced growth suppression by ATP, and are resistant to the effects of β-arrestin-mediated signaling, including PP2-mediated de-phosphorylation of AKT. In patients with the disorder Neurofibromatosis type 1, NF1 mutant SCs proliferate and form SC tumors called neurofibromas. Elevating ATP levels in vivo reduced neurofibroma cell proliferation. Thus, the low proliferation characteristic of differentiated adult peripheral nerve may require ongoing, nerve activity-dependent, ATP. Additionally, we identify a mechanism through which NF1 SCs may evade growth suppression in nerve tumors

Ablating hedgehog signaling in tenocytes during development impairs biomechanics and matrix organization of the adult murine patellar tendon enthesis.

Restoring the native structure of the tendon enthesis, where collagen fibers of the midsubstance are integrated within a fibrocartilaginous structure, is problematic following injury. As current surgical methods fail to restore this region adequately, engineers, biologists, and clinicians are working to understand how this structure forms as a prerequisite to improving repair outcomes. We recently reported on the role of Indian hedgehog (Ihh), a novel enthesis marker, in regulating early postnatal enthesis formation. Here, we investigate how inactivating the Hh pathway in tendon cells affects adult (12‐week) murine patellar tendon (PT) enthesis mechanics, fibrocartilage morphology, and collagen fiber organization. We show that ablating Hh signaling resulted in greater than 100% increased failure insertion strain (0.10 v. 0.05 mm/mm, p<0.01) as well as sub‐failure biomechanical deficiencies. Although collagen fiber orientation appears overtly normal in the midsubstance, ablating Hh signaling reduces mineralized fibrocartilage by 32%, leading to less collagen embedded within mineralized tissue. Ablating Hh signaling also caused collagen fibers to coalesce at the insertion, which may explain in part the increased strains. These results indicate that Ihh signaling plays a critical role in the mineralization process of fibrocartilaginous entheses and may be a novel therapeutic to promote tendon‐to‐bone healing. © 2015 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 33:1142–1151, 2015

The Paratenon Contributes to Scleraxis-Expressing Cells during Patellar Tendon Healing

<div><p>The origin of cells that contribute to tendon healing, specifically extrinsic epitenon/paratenon cells vs. internal tendon fibroblasts, is still debated. The purpose of this study is to determine the location and phenotype of cells that contribute to healing of a central patellar tendon defect injury in the mouse. Normal adult patellar tendon consists of scleraxis-expressing (Scx) tendon fibroblasts situated among aligned collagen fibrils. The tendon body is surrounded by paratenon, which consists of a thin layer of cells that do not express Scx and collagen fibers oriented circumferentially around the tendon. At 3 days following injury, the paratenon thickens as cells within the paratenon proliferate and begin producing tenascin-C and fibromodulin. These cells migrate toward the defect site and express scleraxis and smooth muscle actin alpha by day 7. The thickened paratenon tissue eventually bridges the tendon defect by day 14. Similarly, cells within the periphery of the adjacent tendon struts express these markers and become disorganized. Cells within the defect region show increased expression of fibrillar collagens (Col1a1 and Col3a1) but decreased expression of tenogenic transcription factors (scleraxis and mohawk homeobox) and collagen assembly genes (fibromodulin and decorin). By contrast, early growth response 1 and 2 are upregulated in these tissues along with tenascin-C. These results suggest that paratenon cells, which normally do not express Scx, respond to injury by turning on Scx and assembling matrix to bridge the defect. Future studies are needed to determine the signaling pathways that drive these cells and whether they are capable of producing a functional tendon matrix. Understanding this process may guide tissue engineering strategies in the future by stimulating these cells to improve tendon repair.</p> </div

Paratenon cells produce circumferential collagen fibers as they span the defect space.

<p>The paratenon changes from a thin collagenous structure consisting of 1–2 cell layers of non-Scx-expressing cells (A–B) in normal PT to a thickened structure consisting of circumferential collagen fibers (SHG – blue; white arrowhead) and several layers of green ScxGFP cells (green arrowhead) at day 14 (G–H). As the paratenon cells migrate and bridge the defect space, the tissue is hypercellular and disorganized with a reduced SHG signal (G). PT: patellar tendon, L.R.: lateral retinaculum, M.R.: medial retinaculum; L.S.: lateral strut, M.S.: medial strut. Scale bars are 200 µm in overviews (A, C, E, G) and 100 µm in insets (B, D, F, H).</p

Delta C_T values, relative fold changes (RQ) compared to normal, and principal component 1 (PC1) loadings for each gene of interest

<p>a  =  different than normal, b  =  different than defect/sham at corresponding time point (p<0.05).</p

Scleraxis (Scx) and smooth muscle actin alpha (SMAA) coexpressing cells contribute to tendon healing.

<p>Cells within the thickened paratenon and adjacent struts express both Scx (green) and SMAA (red) following injury. Cells within the paratenon do not express Scx in the normal PT, but smooth muscle cells within blood vessels in the paratenon express SMAA (B–C; red arrows). Regions of high Scx and SMAA expression are within the thickened paratenon and at the anterior and posterior surfaces of the tendon struts (G–L). Scx and SMAA coexpression extends into the interior of the struts with time as seen by the EDM histograms (M). The white arrowheads point to Scx-SMAA coexpressing cells. Error bars indicate ± SD. Scale bars are 200 µm in overviews (A, D, G, J) and 50 µm in insets (B, C, E, F, H, I, K, L).</p

Tendon matrix transitions from predominantly fibromodulin (FMOD) to mixture of FMOD and tenascin-C (TNC).

<p>Cells within the thickened paratenon first express tenascin-C (blue) and fibromodulin (red) on day 3 (D–F) then express scleraxis (green) on days 7 and 14 (G, J). (M) Ratio of FMOD and TNC staining across the tendon width shows that the matrix transitions from close to 95% FMOD in normal tendon to a 50∶50 mixture of FMOD and TNC at day 14. (N) This relationship holds true within the defect region as well. Error bars indicate ± SD. Scale bars are 200 µm in overviews (A, D, G, J) and 50 µm in insets (B, C, E, F, H, I, K, L).</p

Experimental Design

*<p>Twelve animals were pooled together in 3 samples of 4 tendons each for qPCR.</p

Proliferation occurs primarily in non-tenogenic cells outside of tendon.

<p>The EdU labeled cells (yellow) diminish linearly with time while the Ki67 cycling cells (red) remain consistent from days 3 to 7 then reduce at day 14 (M). However, a small subpopulation of cycling cells exists within the activated regions of paratenon and adjacent struts (white arrows), which account for less than 12% of the total cycling cells (N). The plots in the lower panels show quantification of EdU and Ki67 stained nuclei (M) and the number of cycling cells that are Scx+ (N). Error bars indicate ± SD. Scale bars are 200 µm in overviews (A, D, G, J) and 50 µm in insets (B, C, E, F, H, I, K, L).</p

Gene expression of tenogenic transcription factors and fibril assembly proteins were reduced in the defects.

<p>Scx, Mkx, Tnmd, Dcn, and Fmod were all decreased in defects compared to contralateral shams and normal PT. (A) Principal component 1 (PC1) scores (along bottom axis) for treatment groups and loadings for genes of interest (along top axis) show genes that were up-regulated (point to left) and down-regulated (point to the right) in the defects. (B) Scatterplots depicting improved correlations with normal PT over time (horizontally) and changes in expression between the defects and shams (vertically).</p