The α1 integrin cytoplasmic tail interacts with phosphoinositides and interferes with Akt activation.

Integrin α1ß1 is an adhesion receptor that binds to collagen and laminin. It regulates cell adhesion, cytoskeletal organization, and migration. The cytoplasmic tail of the α1 subunit consists of 15 amino acids and contains six positively charged lysine residues. In this study, we present evidence that the α1 integrin cytoplasmic tail (α1CT) directly associates with phosphoinositides, preferentially with phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). Since the association was disrupted by calcium, magnesium and phosphate ions, this interaction appears to be in ionic nature. Here, the peptide-lipid interaction was driven by the conserved KIGFFKR motif. The exchange of both two potential phospholipid-binding lysines for glycines in the KIGFFKR motif increased α1ß1 integrin-specific adhesion and F-actin cytoskeleton formation compared to cells expressing the unmodified α1 subunit, whereas only mutation of the second lysine at position 1171 increased levels of constitutively active α1ß1 integrins on the cell surface. In addition, enhanced focal adhesion formation and increased phosphorylation of focal adhesion kinase, but decreased phosphorylation of AKT was observed in these cells. We conclude that the KIGFFKR motif, and in particular lysine1171 is involved in the dynamic regulation of α1ß1 integrin activity and that the interaction of α1CT with phosphoinositides may contribute to this process.

Proliferation, Migration, and ECM Formation Potential of Human Annulus Fibrosus Cells Is Independent of Degeneration Status.

OBJECTIVE: The objective was to evaluate the proliferating, migratory and extracellular matrix (ECM) forming potential of annulus fibrosus cells derived from early (edAFC) or advanced (adAFC) degenerative tissue and their usability as a possible cell source for regenerative approaches for AF closure. DESIGN: EdAFC (n = 5 Pfirrman score of 2-3) and adAFC (n = 5 Pfirrman score of 4-5) were isolated from tissue of patients undergoing spine stabilizing surgery. Cell migration on stimulation with human serum (HS), platelet-rich plasma (PRP), and transforming growth factor ß-3 (TGFB3) was assessed by migration assay and proliferation was assessed on stimulation with HS. Induction of ECM synthesis was evaluated by gene expression analysis of AF-related genes in three-dimensional scaffold cultures that have been stimulated with 5% PRP or 10 ng/mL TGFB3 and histologically by collagen type I, type II, alcian blue, and safranin-O staining. RESULTS: EdAFC and adAFC were significantly attracted by 10% HS and 5% PRP. Additionally, both cell groups proliferated under stimulation with HS. Stimulation with 10 ng/mL TGFB3 showed significant induction of gene expression of collagen type II and aggrecan, while 5% PRP decreased the expression of collagen type I. Both cell groups showed formation of AF-like ECM after stimulation with TGFB3, whereas stimulation with PRP did not. CONCLUSIONS: Our study demonstrated that AF cells retain their potential for proliferation, migration, and ECM formation independent of the degeneration status of the tissue. Proliferation, migration, and ECM synthesis of the endogenous AF cells can be supported by different supplements. Hence, endogenous AF cells might be a suitable cell source for a regenerative repair approaches.

WISP 1 is an important survival factor in human mesenchymal stromal cells.

WNT-induced secreted protein 1 (WISP1/CCN4), a member of the CCN protein family, acts as a downstream factor of the canonical WNT signaling pathway. Its expression is known to affect proliferation and differentiation of human mesenchymal stromal cells (hMSCs), which are fundamental for the development and maintenance of the musculoskeletal system. Whereas a dysregulated, excessive expression of WISP1 often reflects its oncogenic potential via the inhibition of apoptosis, our study emphasizes the importance of WISP1 signaling for the survival of primary human cells. We have established the efficient and specific down-regulation of endogenous WISP1 transcripts by gene silencing in hMSCs and observed cell death as a consequence of WISP1 deficiency. This was confirmed by Annexin V staining for apoptotic cells. DNA microarray analyses of WISP1 down-regulated versus control samples revealed several clusters of differentially expressed genes important for apoptosis induction such as TNF-related apoptosis-inducing ligand 1 (TRAIL) and the corresponding apoptosis-inducing receptors TRAIL-R1 and -R2. An increased expression of TRAIL and its receptors TRAIL-R1 and -R2 in WISP1-deficient hMSCs was confirmed by immunocytofluorescence. Accordingly, WISP1 deficiency is likely to cause TRAIL-induced apoptosis. This is an important novel finding, which suggests that WISP1 is indispensable for the protection of healthy hMSCs against TRAIL-induced apoptosis.

Human platelet-rich plasma stimulates migration and chondrogenic differentiation of human subchondral progenitor cells.

In cartilage repair, platelet-rich plasma (PRP) is used in one-step approaches utilizing microfracture and matrix-induced chondrogenesis procedures, bone marrow-derived cell transplantation, or intra-articular injection. The aim of our study was to evaluate the effect of human PRP on the migration and chondrogenic differentiation of human subchondral progenitors. Human progenitors were derived from subchondral cortico-spongious bone (CSP), were analyzed for their migration capacity upon PRP treatment in 96-well chemotaxis assays and cultured in high-density pellet cultures under serum-free conditions in the presence of 5% PRP. Chemotaxis assays showed that 0.1-100% PRP significantly (p < 0.05) stimulate the migration of CSP compared to untreated controls. Histological staining of proteoglycan and immuno-staining of type II collagen indicated that progenitors stimulated with PRP show significantly increased cartilage matrix formation compared to untreated progenitors. Real-time gene expression analysis of typical chondrocyte marker genes as well as osteogenic and adipogenic markers like osteocalcin and fatty acid binding protein showed that PRP induces the chondrogenic differentiation sequence of human progenitors in high-density pellet cultures, while osteogenic or adipogenic differentiation was not evident. These results suggest that human PRP may enhance the migration and stimulate the chondrogenic differentiation of human subchondral progenitor cells known from microfracture.