Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway.

Human mesenchymal stem cells (hMSCs) are a population of multipotent bone marrow cells capable of differentiating along multiple lineages, including bone. Our recently published proteomics studies suggest that focusing of gene expression is the basis of hMSC osteogenic transdifferentiation, and that extracellular matrix proteins play an important role in controlling this focusing. Here, we show that application of a 3-5% tensile strain to a collagen I substrate stimulates osteogenesis in the attached hMSCs through gene focusing via a MAP kinase signaling pathway. Mechanical strain increases expression levels of well-established osteogenic marker genes while simultaneously reducing expression levels of marker genes from three alternate lineages (chondrogenic, adipogenic, and neurogenic). Mechanical strain also increases matrix mineralization (a hallmark of osteogenic differentiation) and activation of extracellular signal-related kinase 1/2 (ERK). Addition of the MEK inhibitor PD98059 to reduce ERK activation decreases osteogenic gene expression and matrix mineralization while also blocking strain-induced down-regulation of nonosteogenic lineage marker genes. These results demonstrate that mechanical strain enhances collagen I-induced gene focusing and osteogenic differentiation in hMSCs through the ERK MAP kinase signal transduction pathway.

Comparing the protein expression profiles of human mesenchymal stem cells and human osteoblasts using gene ontologies.

One of the hallmark events regulating the process of osteogenesis is the transition of undifferentiated human mesenchymal stem cells (hMSCs) found in the bone marrow into mineralized-matrix producing osteoblasts (hOSTs) through mechanisms that are not entirely understood. With recent developments in mass spectrometry and its potential application to the systematic definition of the stem cell proteome, proteins that govern cell fate decisions can be identified and tracked during this differentiation process. We hypothesize that protein profiling of hMSCs and hOSTs will identify potential osteogenic marker proteins associated with hMSC commitment and hOST differentiation. To identify markers for each cell population, we analyzed the expression of hMSC proteins and compared them to that of hOST by two-dimensional gel electrophoresis and two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS). The 2D LC-MS/MS data sets were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Only 34% of the spots in 2D gels were found in both cell populations; of those that differed between populations, 65% were unique to hOST cells. Of the 755 different proteins identified by 2D LCMS/ MS in both cell populations, two sets of 247 and 158 proteins were found only in hMSCs and hOST cells, respectively. Differential expression of some of the identified proteins was further confirmed by Western blot analyses. Substantial differences in clusters of proteins responsible for calcium- based signaling and cell adhesion were found between the two cell types. Osteogenic differentiation is accompanied by a substantial change in the overall protein expression profile of hMSCs. This study, using gene ontology analysis, reveals that these changes occur in clusters of functionally related proteins. These proteins may serve as markers for identifying stem cell differentiation into osteogenic fates because they promote differentiation by mechanisms that remain to be defined.

Dissection of the osteogenic effects of laminin-332 utilizing specific LG domains: LG3 induces osteogenic differentiation, but not mineralization.

The overall mechanisms governing the role of laminins during osteogenic differentiation of human mesenchymal stem cells (hMSC) are poorly understood. We previously reported that laminin-332 induces an osteogenic phenotype in hMSC and does so through a focal adhesion kinase (FAK) and extracellular signal-related kinase (ERK) dependent pathway. We hypothesized that this is a result of integrin-ECM binding, and that it occurs via the known alpha3 LG3 integrin binding domain of laminin-332. To test this hypothesis we cultured hMSC on several different globular domains of laminin-332. hMSC adhered best to the LG3 domain, and this adhesion maximally activated FAK and ERK within 120 min. Prolonged culturing (8 or 16 days) of hMSC on LG3 led to activation of the osteogenic transcription factor Runx2 and expression of key osteogenic markers (osterix, bone sialoprotein 2, osteocalcin, alkaline phosphatase, extracellular calcium) in hMSC. LG3 domain binding did not increase matrix mineralization, demonstrating that the LG3 domain alone is not sufficient to induce complete osteogenic differentiation in vitro. We conclude that the LG3 domain mediates attachment of hMSC to laminin-332 and that this adhesion recapitulates most, but not all, of the osteogenic differentiation associated with laminin-5 binding to hMSC.

Focal adhesion kinase signaling controls cyclic tensile strain enhanced collagen I-induced osteogenic differentiation of human mesenchymal stem cells.

Focal adhesion kinase (FAK) is a key integrator of integrin-mediated signals from the extracellular matrix to the cytoskeleton and downstream signaling molecules. FAK is activated by phosphorylation at specific tyrosine residues, which then stimulate downstream signaling including the ERK1/2 pathway, leading to a variety of cellular responses. In this study, we examined the effects of FAK point mutations at tyrosine residues (Y397, Y925, Y861, and Y576/7) on osteogenic differentiation of human mesenchymal stem cells exposed to collagen I and cyclic tensile strain. Our results demonstrate that FAK signaling emanating from Y397, Y925, and to a lesser extent Y576/7, but not from Y861, controls osteogenic differentiation through an ERK1/2 pathway, as measured by expression levels of key osteogenesis marker genes and subsequent matrix mineralization. These data indicate that FAK is a critical decision maker in extracellular matrix/strain-enhanced osteogenic differentiation.