Laminin-5 induces osteogenic gene expression in human mesenchymal stem cells through an ERK-dependent pathway

Abstract

The laminin family of proteins is critical for managing a variety of cellular activities including migration, adhesion, and differentiation. In bone, the roles of laminins in controlling osteogenic differentiation of human mesenchymal stem cells (hMSC) are unknown. We report here that laminin-5 is found in bone and expressed by hMSC. hMSC isolated from bone synthesize laminin-5 and adhere to exogenous laminin-5 through alpha3beta1 integrin. Adhesion to laminin-5 activates extracellular signal-related kinase (ERK) within 30 min and leads to phosphorylation of the osteogenic transcription factor Runx2/CBFA-1 within 8 d. Cells plated on laminin-5 for 16 d express increased levels of osteogenic marker genes, and those plated for 21 d deposit a mineralized matrix, indicative of osteogenic differentiation. Addition of the ERK inhibitor PD98059 mitigates these effects. We conclude that contact with laminin-5 is sufficient to activate ERK and to stimulate osteogenic differentiation in hMSC.

Figure 1.

Immunolocalization of Ln-5 subunits to the periosteum of rat rib. Enzyme-linked fluorescence technology was used to detect immunostaining (arrows) of Ln α3 and γ2 chains with CM6 and TR1 antibodies in frozen sections of adult rat rib (E and F, respectively). Controls include autofluorescence (A), reduced autofluorescence following exposure to UV light (B, bone-cartilage junction; and D, rib synovium), background staining from uv-treated section stained with normal mouse IgG (C). All images taken at 10× magnification. Cortical bone shattered during frozen sectioning.

Figure 2.

hMSC express Ln-5 in culture. (A–D) Cells were grown on glass coverslips for 8 d and then were fixed and stained for α3 integrin (A and C) and human Ln-5 (B and D). (A) Magnification, 40×. (B–D) Magnification, 10×. Images in C and D were taken of the same microscopic field. (E) RT-PCR was used to amplify the indicated mRNA transcripts from hMSC grown on tissue culture plastic. The α1 and β1 chains, found in Ln-1, were included as a positive control. GAPDH was amplified as a positive control.

Figure 3.

hMSC bind to and migrate on Ln-5. (A) Static 30-min assay of hMSC adhesion to purified ECM proteins. Adherent cells were stained with crystal violet and then solubilized in SDS, and absorbance was determined at 595 nm. Values represent mean ± SD (n = 5). (B) Cells were plated in the upper chamber of a MIC filter migration plate (Millipore) and allowed to migrate toward the lower chamber through a 8-μm filter coated with Ln-5. Controls had filters coated with COLL-I, VN, FN, or nd-blotto. Each condition was repeated in 12 wells.

Figure 4.

hMSC use α3β1 integrin to bind Ln-5. Adhesion assays as per Figure 3A were performed in the presence of integrin blocking antibodies.

Figure 5.

Adhesion to Ln-5 activates ERK in hMSC. (A) Cells kept in suspension (susp.), or plated for 30 min on poly-l-lysine (Poly-L-L), tissue culture plastic with OS media (+OS), or on Ln-5 were probed by immunoblot for phosphorylated ERK (top row), total ERK (middle row), or actin as a loading control (bottom row). Where indicated by a “+” symbol, 50 μM PD98059 was added to the culture conditions. (B and C) Densitometric measurements of band intensities for phospho-ERK 1 and phospho-ERK 2, respectively.

Figure 6.

Adhesion to Ln-5 stimulates runx2/CBFA-1 phosphorylation. Cells were plated for 1 d (top row) or 8 d (middle and bottom rows) on tissue culture plastic in the absence (CTL) or presence of OS media (+OS), or on Ln-5, and then lysed, and runx2/CBFA-1 was immunoprecipitated. The immunoprecipitated proteins were separated by SDS-PAGE and probed by immunoblot for phosphoserine (top two rows) or total runx2/CBFA-1 as a loading control (bottom row). Where indicated by a “+” symbol, 50 μM PD98059 was added to the culture conditions. (B and C) Densitometric measurements of bands for pRunx2 on days 1 and 8, respectively.

Figure 7.

Ln-5 induces expression of osteopontin and osteocalcin in hMSC. (A) RT-PCR was performed on hMSC grown for 16 d on tissue culture plastic in the absence (CTL) or presence of OS media (+OS), or on Ln-5. Where indicated by a “+” symbol, 50 μM PD98059 was added to the culture conditions. Indicated genes were amplified using primers listed in Table 1. GAPDH was amplified as a loading control. (B–E) Densitometric analysis of bands in A for (B) ALP, (C) osteocalcin, (D) osteopontin, and (E) runx2/CBFA-1.

Figure 8.

hMSC plated on Ln-5 express osteocalcin and osteopontin. Cells were plated for 16 d under the same conditions as in Figure 7 and probed by immunoblot for osteocalcin (OC) and osteopontin (OP).

Figure 9.

Ln-5 induces matrix mineralization. FTIR analysis of hMSC plated on tissue culture plastic (A) or Ln-5 (B) for 21 d. The mineral:matrix is computed by comparing the area of the phosphate peak (mineral) to the amide peak (protein) shown.

Figure 10.

Fibronectin is not an osteogenic stimulus for hMSC. (A) hMSC were plated on poly-l-lysine, fibronectin, and collagen-I for 30 and 60 min and assayed for ERK activation as in Figure 5. (B) Calcium deposition of hMSC plated on poly-l-lysine, fibronectin, and collagen-I for 16 d. Calcium was solublized in HCl, and absorbance was determined at 575 nm. Values represent mean ± SD (n = 4).