Extracellular matrix protein tenascin-C is required in the bone marrow microenvironment primed for hematopoietic regeneration

Abstract

The BM microenvironment is required for the maintenance, proliferation, and mobilization of hematopoietic stem and progenitor cells (HSPCs), both during steady-state conditions and hematopoietic recovery after myeloablation. The ECM meshwork has long been recognized as a major anatomical component of the BM microenvironment; however, the molecular signatures and functions of the ECM to support HSPCs are poorly understood. Of the many ECM proteins, the expression of tenascin-C (TN-C) was found to be dramatically up-regulated during hematopoietic recovery after myeloablation. The TN-C gene was predominantly expressed in stromal cells and endothelial cells, known as BM niche cells, supporting the function of HSPCs. Mice lacking TN-C (TN-C(-/-)) mice showed normal steady-state hematopoiesis; however, they failed to reconstitute hematopoiesis after BM ablation and showed high lethality. The capacity to support transplanted wild-type hematopoietic cells to regenerate hematopoiesis was reduced in TN-C(-/-) recipient mice. In vitro culture on a TN-C substratum promoted the proliferation of HSPCs in an integrin α9-dependent manner and up-regulated the expression of the cyclins (cyclinD1 and cyclinE1) and down-regulated the expression of the cyclin-dependent kinase inhibitors (p57(Kip2), p21(Cip1), p16(Ink4a)). These results identify TN-C as a critical component of the BM microenvironment that is required for hematopoietic regeneration.

Figure 1

TN-C is markedly up-regulated in the BM during myeloablation and hematopoietic recovery. BM sections from days 0, 2, and 10 stained for TN-C (red) in the metaphyseal (A-C) and diaphyseal (D-F) regions of the femoral bone. The bone surfaces are outlined by white dotted lines (A-F). The asterisks in panels C and F show the up-regulation of TN-C beyond the endosteal areas in both the metaphyseal and diaphyseal regions. (G-H) Enlargement of the dotted squares in panels D and F. Arrows indicate the bone surface; arrowheads indicate stromal TN-C expression. (I) High magnification of day 10 BM samples stained for TN-C (red), c-Kit (green), laminin (blue), and DAPI (gray). c-Kit⁺ cells adhered to TN-C expressed perivascularly (costained with laminin; open arrowheads) or away from the vasculature (arrowheads). (J) Western blotting for TN-C in total BM proteins showed that TN-C proteins were detected in 2 bands at 280 and 220 kDa. (K) Quantification of Western blot expression. The mean fluorescence ratio was calculated by dividing the mean fluorescence of the TN-C band by that of the corresponding β-actin band. *P < .05. Scale bars indicate 200 μm in panels A through F; 40 μm in panels G and H; and 10 μm in panel I.

Figure 2

TN-C is predominantly expressed in stromal and endothelial cells and its ligand, integrin α9, is expressed on HSPCs. (A-B) Relative RT-PCR for TN-C mRNA expression on whole-BM (A) and on CD45⁺, CD11b⁺, CD31⁺CD45⁻, PDGFRα⁺CD45⁻, and LSK cells (B; n = 5). (C-E) IHC of BM obtained from Cxcl12^+/EGFP mice on day 10. Note that TN-C proteins (red) are deposited around CD31⁺ (blue) endothelial cells (arrows) or CAR cells (green; arrowheads). (F) Relative expression of TN-C mRNA in isolated CD45⁻Ter119⁻CD31⁺Sca1⁺ (ECs), Cxcl12^neg and Cxcl12^high cells on day 10 (n = 5). (G-N) Relative expressions of integrin α9 (G), integrin β1 (H), integrin α4 (I), integrin αV (J), integrin α5 (K), integrin α7 (L), integrin β3 (M), and integrin β6 (N) in whole BM cells (n = 6). (O) Flow cytometric analysis of integrin α9 and integrin β1 expression by LSK cells. (P) Ratio (Lin⁺ = 1) of mean fluorescence for integrin α9 and integrin β1 in the Lin⁺ and LSK fractions (n = 7). *P < .05. Scale bar indicates 50 μm.

Figure 3

Normal steady-state hematopoiesis in TN-C^−/− mice. (A-C) FACS plots showing BM lineage frequency. (D) Quantification in each lineage (n = 4). (E) FACS plot showing the detection of LSK cells. (F-G) Total MNC count (F) and LSK cell count (G; n = 4). (H-K) FACS plots (H-I) and quantification (n = 4; J-K) of Flt3⁻CD34⁻ LSK and CD48⁻CD41⁻CD150⁺ LSK cell frequency. (L-M) Cell-cycle status frequencies assessed through Ki-67 and Hoechst staining of LSK cells (n = 6). *P < .05.

Figure 4

TN-C^−/− mice show impaired hematopoietic recovery after 5-FU administration. (A-B) IHC for TN-C (red) and DAPI (blue) for day 7 BMs. TN-C^−/− mice lack TN-C immunoreactivity. (C) Survival curve for TN-C^+/+ and TN-C^−/− mice after 5-FU administration (n = 12; combination of 3 independent experiments). (D-F) Peripheral blood counts for WBCs (D), hemoglobin (Hb; E), and platelets (Plt; F) after 5-FU administration. (G) BM lineage frequencies on day 10 after 5-FU administration (n = 4). (H-I) IHC for c-Kit (green) and DAPI (blue) in TN-C^+/+ and TN-C^−/− BM on day 10 after 5-FU administration. (J-K) IHC for BrdU (red) and c-Kit (green) in TN-C^+/+ and TN-C^−/− BM on day 10 after 5-FU administration. (L) Quantification of the percentage of c-Kit⁺ cells among DAPI⁺ BM cells (n = 6). (M) Quantification of the percentage of BrdU⁺ cells among c-Kit⁺ cells (n = 6). (N-O) FACS plot and quantification showing the detection of LSK cells on day 10 after 5-FU administration (n = 4). (P) Survival curves for TN-C^+/+ and TN-C^−/− mice subjected to 6.5 Gy irradiation (n = 5; combination of 3 independent experiments). *P < .05. Scale bars indicate 100 μm in panels A, B, H, and I and 20 μm in panels J and K.

Figure 5

TN-C^−/− mice are vulnerable recipients for BMT. (A) Peripheral blood chimerism analyses of recipient mice infused with 2 × 10⁵ donor cells from TN-C^+/+ or TN-C^−/− mice (n = 10). (B-D) BMMNC count (B), LSK cell frequency (C), and LSK cell chimerism (D) of recipient mice 4 months after transplantation with 2 × 10⁵ donor cells from TN-C^+/+ and TN-C^−/− mice (n = 4). (E) Survival curves for TN-C^+/+ and TN-C^−/− recipient mice infused with 2 × 10⁵ cells (white and black boxes, respectively) or 1 × 10⁴ cells (white and black circles, respectively; n > 10 for each group; combination of 3 independent experiments). (F-G) Flow cytometric analysis of the BM in TN-C^+/+ or TN-C^−/− recipient mice 4 months after BMT (2 × 10⁵ cells) showing the frequency and chimerism of Flt3⁻CD34⁻ LSK cells and CD48⁻CD41⁻CD150⁺ LSK cells (n = 4). (H-I) BM lineage composition (H) and chimerism (I) of cells within each lineage in the BM of TN-C^+/+ or TN-C^−/− recipient mice 4 months after BMT (2 × 10⁵ cells; n = 4). (J) Survival curves for TN-C^+/+ and TN-C^−/− recipient mice challenged with 5-FU 4 months after BMT (2 × 10⁵ cells; n = 10; combination of 3 independent experiments). (K-M) Peripheral blood counts of mice shown in panel J (n = 10). *P < .05.

Figure 6

TN-C coating enhances the in vitro proliferation and expression of cell-cycle–promoting genes in HSPCs in an integrin α9–dependent manner. (A-E) Short-term BrdU incorporation assay for LSK cells cultured for 48 hours on slides coated with FN alone, FN plus full-length TN-C (FN + TN-C), or FN plus TN-C^FNIII (FN + TN-C^FNIII) with functional integrin α9 Abs (55A2C) or nonfunctional integrin α9 Abs (18R18D). (F) Percentage of BrdU⁺LSK cells in panels A-E (n = 4). (G) Relative mRNA expression for the cell-cycle regulators c-myc, cyclinD1, cyclinD2, cyclinD3, cyclinE1, cyclinE2, cyclinG1, cyclinG2, p57^Kip2, p18^Ink4c, p21^Cip1, and p16^Ink4a (n = 4). *P < .05.