A role for TGFβ signaling in Gli1+ tendon and enthesis cells

Abstract

The development of musculoskeletal tissues such as tendon, enthesis, and bone relies on proliferation and differentiation of mesenchymal progenitor cells. Gli1+ cells have been described as putative stem cells in several tissues and are presumed to play critical roles in tissue formation and maintenance. For example, the enthesis, a fibrocartilage tissue that connects tendon to bone, is mineralized postnatally by a pool of Gli1+ progenitor cells. These cells are regulated by hedgehog signaling, but it is unclear if TGFβ signaling, necessary for tenogenesis, also plays a role in their behavior. To examine the role of TGFβ signaling in Gli1+ cell function, the receptor for TGFβ, TbR2, was deleted in Gli1-lineage cells in mice at P5. Decreased TGFβ signaling in these cells led to defects in tendon enthesis formation by P56, including defective bone morphometry underlying the enthesis and decreased mechanical properties. Immunohistochemical staining of these Gli1+ cells showed that loss of TGFβ signaling reduced proliferation and increased apoptosis. In vitro experiments using Gli1+ cells isolated from mouse tail tendons demonstrated that TGFβ controls cell proliferation and differentiation through canonical and non-canonical pathways and that TGFβ directly controls the tendon transcription factor scleraxis by binding to its distant enhancer. These results have implications in the development of treatments for tendon and enthesis pathologies.

Keywords: Gli1; TGFβ; TbR2; development; enthesis.

Figure 1.. Generation and validation of Gli1Cre^ERT2;TbR2^fl/fl;mTmG mice.

(A) Strategy for generating reporter mice whose Gli1-lineage TbR2-deleted cells were tagged with eGFP. (B) Immunocytochemistry of cultured cells showed that TGFβ-induced Smad2 nuclear localization was blocked in Gli1-lineage (green) cells with TbR2 deletion (top row), in contrast to non-Gli1-lineage cells (red) (bottom row). (C) Western blot analysis of cultured cells demonstrated substantially reduced Smad2 phosphorylation in Gli1Cre^ERT2;TbR2^fl/fl cells (CKO) cells compared to control cells.

Figure 2.. TbR2 deletion in Gli1-lineage cells led to reduced enthesis mechanical properties and altered bone morphometry.

(A) Tamoxifen (TAM) was administrated at P5 and P7 to induce deletion of TbR2 in Gli1-expressing cells (CKO). (B) The mechanical properties of the supraspinatus tendon enthesis were reduced in CKO mice compared to CTRL mice. * p<0.05, ** p<0.01, *** p<0.001. (C) Deletion of TbR2 in Gli1-expressing cells led to altered bone morphometry of the humeral head cortical and trabecular bone. (D) Representative histologic sections for control and CKO supraspinatus tendon entheses (Safranan O stain, tendon is on the left and bone is on the right of the sections; scale bars = 50 μm). (E) Representative microCT images show reduced trabecular bone and lower bone mineral density adjacent to the supraspinatus tendon enthesis (arrows). ** p<0.01, *** p<0.001.

Figure 3.. ECM changes in Gli1Cre^ERT2;TbR2^fl/fl CKO mice were limited to Gli1-lineage cells.

Immunohistochemistry for (A) COL1a (magenta) and (B) SPARC (red) in Control and CKO supraspinatus tendon entheses. Gli1-lineage cells are GFP+ (green) (bone is on the left and tendon is on the right of the sections; scale bars = 50 μm). The insert is an enlarged view of the white boxed area of the enthesis (positive cells indicated by arrowheads). Histomophometric analyses demonstrated numbers and percentage of cells positive for (A) COL1a and (B) SPARC for the enthesis and for Gli1-lineage cells (i.e., colocalization of GFP with either COL1a or SPARC). * p<0.05.

Figure 4.. TbR2 deletion led to fewer Gli1-lineage cells.

(A) FACS plots and quantification showed reduced numbers of Gli1-lineage cells in CKO mice during cell culture (passage 3). (B) Ki67 immunostaining revealed Ki67+ in Gli1-lineage cells in control and CKO supraspinatus entheses. Arrows indicate Ki67+ Gli1-lineage cells (tendon is on the left and bone is on the right of the sections). (C) TUNEL staining revealed apoptotic cells in Gli1-lineage cells in control and CKO supraspinatus enthesis. Arrows indicate TUNEL-positive Gli1-lineage cells (tendon is on the right and bone is on the left of the sections) (scale bars=50μm). ns: not significant, * p<0.05, *** p<0.001.

Figure 5.. Responses of Gl1-lineage cells to TGFβ and HhAg in vitro.

Quantification of Scx and Sox9 expression by qPCR in cultured Gli1-lineage cells between control and CKO mice. Left panel: cells treated with TGFβ, right panel: cells treated with hedgehog agonist (HhAg). TbR2 deletion impaired TGFβ-induced Scx expression and HhAg-induced Sox9 expression in Gli1-lineage cultured cells. Statistically significant effects of genotype and time were determined by two factor ANOVA.

Figure 6.. TGFβ induced non-canonical pathways in Gli1-lineage cells.

(A) Representative western blots for control and CKO Gli1-lineage cells. (B) Quantification of phosphorylated cJun, JNK, p38, and Erk in control and CKO Gli1-lineage cells. Statistical significance for the effect of genotype and time was determined by two factor ANOVA. (C) Immunofluorescent staining of p-cJun (magenta) in Control and CKO supraspinatus tendon entheses. Gli1-lineage cells are GFP+ (green) (bone is on the lower left and tendon is on the upper left of the sections; positive cells indicated by arrowheads; scale bars = 50 μm.

Figure 7.. Identification of Smad4 binding site on Scx enhancer.

(A) Three potential Smad4 binding sites in the mouse Scx locus, as predicted by Promo software. (B) Time-dependent Smad4 binding to 3 potential sites. (C) Comparison of TGFβ induced Smad4 binding to 4.9 kb Scx enhancer between control and CKO cells.