Fibroblast activation protein (FAP) is essential for the migration of bone marrow mesenchymal stem cells through RhoA activation.

BACKGROUND: The ability of human bone marrow mesenchymal stem cells (BM-MSCs) to migrate and localize specifically to injured tissues is central in developing therapeutic strategies for tissue repair and regeneration. Fibroblast activation protein (FAP) is a cell surface serine protease expressed at sites of tissue remodeling during embryonic development. It is also expressed in BM-MSCs, but not in normal tissues or cells. The function of FAP in BM-MSCs is not known. PRINCIPAL FINDINGS: We found that depletion of FAP proteins significantly inhibited the migration of BM-MSCs in a transwell chemotaxis assay. Such impaired migration ability of BM-MSCs could be rescued by re-expressing FAP in these cells. We then demonstrated that depletion of FAP activated intracellular RhoA GTPase. Consistently, inhibition of RhoA activity using a RhoA inhibitor rescued its migration ability. Inhibition of FAP activity with an FAP-specific inhibitor did not affect the activation of RhoA or the migration of BM-MSCs. Furthermore, the inflammatory cytokines interleukin-1beta (IL-1β) and transforming growth factor-beta (TGF-β) upregulated FAP expression, which coincided with better BM-MSC migration. CONCLUSIONS: Our results indicate FAP plays an important role in the migration of BM-MSCs through modulation of RhoA GTPase activity. The peptidase activity of FAP is not essential for such migration. Cytokines IL-1β and TGF-β upregulate the expression level of FAP and thus enhance BM-MSC migration

Rho GTPase activation in FAP-depleted BM-MSCs.

<p>(A) The amount of GTP-bound RhoA was determined with the cells depleted of FAP, as described in the Materials and methods section. The amounts of GTP-bound RhoA were normalized against total RhoA protein present in cell lysates and expressed as fold induction compared with the cells infected with the vector control. Data are representative of two independent experiments. (B) Results are shown as in (A) except that GTP-bound Rac1 was used. Data are representative of two independent experiments. (C) Effect of RhoA inhibitors on the migration of FAP-depleted BM-MSCs was determined by transwell assays. FAP-depleted BM-MSCs were pretreated without or with the RhoA inhibitor C2I-C3 (1 µg/ml) (Cytoskeleton Inc.) in IMDM for 2 h and seeded in the upper wells of the chambers. The lower wells contained IMDM with 10% FBS. Cells were allowed to migrate for 48 h and absolute cell numbers were determined as described in the Materials and methods section. Data are representative of three separate experiments each performed in triplicate. Means and standard deviations were calculated. The error bars are showing standard deviation (SD). The statistically significant differences between the groups were assessed using a two-tailed Student's t test. The degree of significance is indicated as fellows: **p<0.001; ***p<0.0001.</p

FAP depletion in BM-MSCs by lentiviral infection.

<p>(A) BM-MSCs were depleted of FAP by transfection with lentiviruses encoding two FAP shRNA sequences. The efficiency of shRNA-based downregulation was determined by qRT–PCR. Results are given as the percentage of change in mRNA expression relative to pLKO empty vector-infected cells set as 100%. The error bars are showing standard deviation (SD). (B) The efficiency of shRNA-based downregulation was determined by western blot analysis. Data are representative of three independent experiments. (C–F) Flow cytometric analysis of cell surface markers on FAP-depleted BM-MSCs. (C) Parental BM-MSCs were analyzed using antibodies against CD34, CD73, CD90, and CD105. (D) Vector infected BM-MSCs were analyzed using antibodies against CD34, CD73, CD90, and CD105. (E) As in (D) but with FAP shRNA-A infected BM-MSCs. (F) As in (D) but with FAP shRNA-B infected BM-MSCs.</p