Mechanism of Divergent Growth Factor Effects in Mesenchymal Stem Cell Differentiation

Kratchmarova, Irina; Blagoev, Blagoy; Haack‐Sørensen, Mandana; Kassem, Moustapha; Mann, Matthias

doi:10.1126/science.1107627

Cited by 530 publications

(428 citation statements)

References 31 publications

Supporting

Mentioning

408

Contrasting

Unclassified

Order By: Relevance

“…In other cells, these FAK complexes are sensitive to a wide variety of extracellular stimuli beyond ECM binding, including glucocorticoids [36], peptide growth factors [37], and mechanical stress [38]. These same stimuli contribute to osteogenic differentiation on hMSC [5,39,40], and our data support the idea that FAK mediates this response.…”

Section: Discussionsupporting

confidence: 81%

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

Salasznyk

Klees

Williams

et al. 2007

Experimental Cell Research

263

225

View full text Add to dashboard Cite

The intracellular signaling events controlling human mesenchymal stem cell (hMSC) differentiation into osteoblasts are not entirely understood. We recently demonstrated that contact with extracellular matrix (ECM) proteins is sufficient to induce osteogenic differentiation of hMSC through an ERKdependent pathway. We hypothesized that FAK signaling pathways provide a link between activation of ERK 1/2 by ECM, and stimulate subsequent phosphorylation of the Runx2/Cbfa-1 transcription factor that controls osteogenic gene expression. We plated hMSC on purified collagen I (COLL-I) and vitronectin (VN) in the presence or absence of FAK-specific siRNA, and assayed for phosphorylation of Runx2/Cbfa-1 as well as expression of established osteogenic differentiation markers (bone sialoprotein-2, osteocalcin, alkaline phosphatase, calcium deposition, and spectroscopically determined mineral:matrix ratio). We found that siRNA treatment reduced FAK mRNA levels by >40% and decreased ECM-mediated phosphorylation of FAK Y397 and ERK 1/2. Serine phosphorylation of Runx2/Cbfa-1 was significantly reduced after 8 days in treated cells. Finally, FAK inhibition blocked osterix transcriptional activity and the osteogenic differentiation of hMSC, as assessed by lowered expression of osteogenic genes (RT-PCR), decreased alkaline phosphatase activity, greatly reduced calcium deposition, and a lower mineral:matrix ratio after 28 days in culture. These results suggest that FAK signaling plays an important role in regulating ECMinduced osteogenic differentiation of hMSC.

show abstract

Section: Discussionsupporting

confidence: 81%

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

Salasznyk

Klees

Williams

et al. 2007

Experimental Cell Research

263

225

View full text Add to dashboard Cite

show abstract

“…In particular, two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) is a powerful approach for identifying protein constituents in cell populations. For example, mass spectrometry reveals differences in tyrosine phosphorylation of hMSC proteins in response to epidermal growth factor (EGF), plateletderived growth factor (PDGF), and transforming growth factor β1 (TGFβ1) (Wang et al, 2004;Kratchmarova et al, 2005), and suggests that phosphatidylinositol 3-kinase is a possible control point in the osteogenic differentiation process. Mass spectroscopic profiling of proteins expressed in ECM-stimulated hMSC has thus far not been reported.…”

Section: Introductionmentioning

confidence: 99%

Laminin-5 activates extracellular matrix production and osteogenic gene focusing in human mesenchymal stem cells

et al. 2007

View full text Add to dashboard Cite

We recently reported that laminin-5, expressed by human mesenchymal stem cells (hMSC), stimulates osteogenic gene expression in these cells in the absence of any other osteogenic stimulus. Here we employ two dimensional liquid chromatography and tandem mass spectrometry, along with the Database for Annotation, Visualization and Integrated Discovery (DAVID), to obtain a more comprehensive profile of the protein (and hence gene) expression changes occurring during laminin-5-induced osteogenesis of hMSC. Specifically, we compare the protein expression profiles of undifferentiated hMSC, hMSC cultured on laminin-5 (Ln-5 hMSC), and fully differentiated human osteoblasts (hOST) with profiles from hMSC treated with well-established osteogenic stimuli (collagen I, vitronectin, or dexamethazone). We find a marked reduction in the number of proteins (e.g., those involved with calcium signaling and cellular metabolism) expressed in Ln-5 hMSC compared to hMSC, consistent with our previous finding that hOST express far fewer proteins than do their hMSC progenitors, a pattern we call "osteogenic gene focusing." This focused set, which resembles an intermediate stage between hMSC and mature hOST, mirrors the expression profiles of hMSC exposed to established osteogenic stimuli and includes osteogenic extracellular matrix proteins (collagen, vitronectin) and their integrin receptors, calcium signaling proteins, and enzymes involved in lipid metabolism. These results provide direct evidence that laminin-5 alone stimulates global changes in gene/protein expression in hMSC that lead to commitment of these cells to the osteogenic phenotype, and that this commitment correlates with extracellular matrix production.

show abstract

“…The complete set of residue composition vectors can be assembled into a residue composition matrix: (24) The residue formula is given by a vector p̂ of length n residues , where the elements specify the number of each residue type in the species. The relationship between the molecular formula and residue formula is given by: (25) The μ-domain representation f(μ) can then be represented in the alternative residue basis (26) where (27) Essentially the expressions in eqs 26 and 27 factor the μ-domain function in eq 4 according to residues instead of atoms. This new form allows for the incorporation of fractional residue labeling in calculation of isotope distributions.…”

Section: Residue-based Convolution Of Isotope Distributionsmentioning

confidence: 99%

Quantitative Analysis of Isotope Distributions In Proteomic Mass Spectrometry Using Least-Squares Fourier Transform Convolution

et al. 2008

View full text Add to dashboard Cite

Quantitative proteomic mass spectrometry involves comparison of the amplitudes of peaks resulting from different isotope labeling patterns, including fractional atomic labeling and fractional residue labeling. We have developed a general and flexible analytical treatment of the complex isotope distributions that arise in these experiments, using Fourier transform convolution to calculate labeled isotope distributions and least-squares for quantitative comparison with experimental peaks. The degree of fractional atomic and fractional residue labeling can be determined from experimental peaks at the same time as the integrated intensity of all of the isotopomers in the isotope distribution. The approach is illustrated using data with fractional 15 N-labeling and fractional 13 C-isoleucine labeling. The least-squares Fourier transform convolution approach can be applied to many types of quantitive proteomic data, including data from stable isotope labeling by amino acids in cell culture and pulse labeling experiments.Stable isotope labeling in cells coupled with mass spectrometry has many important applications in the analysis of protein expression, modification, turnover, and metabolism. 1 Cells and organisms can be isotopically labeled by supplying labeled precursors in the form of nutrients such as amino acids, glucose, and ammonia. These labeled precursors are incorporated into proteins, whose resulting isotope labeling patterns reflect their abundance and the dynamics of protein synthesis and turnover. The era of quantitative proteomics began with experiments where mixtures of unlabeled control cells and 15 N-labeled or isotope depleted test cells were quantitatively analyzed to determine protein expression and phosphorylation levels. 2,3 More recently, the SILAC technique was developed based on specific amino acid labeling of cells for quantitative analysis of protein expression, modification, and turnover. 4-6The two basic classes of quantitative metabolic labeling approaches are the stable isotope labeling by amino acids in cell culture (SILAC) experiments and pulse labeling experiments, both of which have been implemented in a variety of ways. 7 In the SILAC method, independent samples are prepared with different labeling patterns that are mixed prior to mass spectrometry analysis. For pulse labeling experiments, labels that are added to the growth medium are taken up to metabolically label the proteome, which generates fractionally enriched proteins from a

show abstract

Mechanism of Divergent Growth Factor Effects in Mesenchymal Stem Cell Differentiation

Cited by 530 publications

References 31 publications

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

Laminin-5 activates extracellular matrix production and osteogenic gene focusing in human mesenchymal stem cells

Quantitative Analysis of Isotope Distributions In Proteomic Mass Spectrometry Using Least-Squares Fourier Transform Convolution

Contact Info

Product

Resources

About