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

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.     

A novel collagen-binding peptide promotes osteogenic differentiation via Ca2+/calmodulin-dependent protein kinase II/ERK/AP-1 signaling pathway in human bone marrow-derived mesenchymal stem cells

The intracellular signaling events controlling human mesenchymal stem cell (hMSC) differentiation into osteoblasts are poorly understood. Collagen-binding domain is considered an essential component of bone mineralization. In the present study, we investigated the regulatory mechanism of osteoblastic differentiation of hMSC by the peptide with a novel collagen-binding motif derived from osteopontin. The peptide induced influx of extracellular Ca2+ via calcium channels and increased intracellular Ca2+ concentration ([Ca2+]i) independent of both pertussis toxin and phospholipase C, and activated ERK, which was inhibited by Ca2+/calmodulin-dependent protein kinase (CaMKII) antagonist, KN93. The peptide-induced increase of [Ca2+]i is correlated with ERK activation in a various cell types. The peptide stimulated the migration of hMSC but suppressed cell proliferation. Furthermore, the peptide increased the phosphorylation of cAMP-response element-binding protein, leading to a significant increase in the transactivation of cAMP-response element and serum response element. Ultimately, the peptide increased AP-1 transactivation, c-jun expression, and bone mineralization, which are suppressed by KN93. Taken together, these results indicate that the novel collagen-binding peptide promotes osteogenic differentiation via Ca2+/CaMKII/ERK/AP-1 signaling pathway in hMSC, suggesting the potential application in cell therapy for bone regeneration.     

Laminin-1 induces neurite outgrowth in human mesenchymal stem cells in serum/differentiation factors-free conditions through activation of FAK-MEK/ERK signaling pathways

Mesenchymal stem cells (MSCs) can be differentiated into cell types derived from all three germ layers by manipulating culture conditions in vitro. A multitude of growth and differentiation factors have been employed for driving MSCs towards a neuronal phenotype. In the present study, we investigated the potential of extracellular matrix (ECM) proteins—fibronectin, collagen-1, collagen-IV, laminin-1, and laminin-10/11, to induce a neuronal phenotype in bone marrow derived human MSCs in the absence of growth factors/differentiating agents. All of the ECM proteins tested were found to support adhesion of MSCs to different extents. However, direct interaction only with laminin-1 triggered sprouting of neurite-like processes. Cells plated on laminin-1 exhibited neurite out growth as early as 3 h, and by 24 h, the cells developed elaborate neurites with contracted cell bodies and neuronal-like morphology. Function-blocking antibodies directed against α6 and β1 integrin subunits inhibited neurite formation on laminin-1 which confirmed the involvement of integrin α6β1 in neurite outgrowth. Mechanistic studies revealed that cell adhesion to laminin-1 activated focal adhesion kinase (FAK), and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways. Abrogation of FAK phosphorylation by herbimycin-A inhibited neurite formation and also decreased activities of MEK and ERK. Pharmacological inhibitors of MEK (U0126) and ERK (PD98059) also blocked neurite outgrowth in cells plated on laminin-1. Our study demonstrates the involvement of integrin α6β1 and FAK-MEK/ERK signaling pathways in laminin-1-induced neurite outgrowth in MSCs in the absence of serum and differentiation factors.     

The Casitas B lineage lymphoma (Cbl) mutant G306E enhances osteogenic differentiation in human mesenchymal stromal cells in part by decreased Cbl-mediated platelet-derived growth factor receptor alpha and fibroblast growth factor receptor 2 ubiquitination

Human bone marrow-derived mesenchymal stromal cells (hMSCs) have the capacity to differentiate into several cell types including osteoblasts and are therefore an important cell source for bone tissue regeneration. A crucial issue is to identify mechanisms that trigger hMSC osteoblast differentiation to promote osteogenic potential. Casitas B lineage lymphoma (Cbl) is an E3 ubiquitin ligase that ubiquitinates and targets several molecules for degradation. We hypothesized that attenuation of Cbl-mediated degradation of receptor tyrosine kinases (RTKs) may promote osteogenic differentiation in hMSCs. We show here that specific inhibition of Cbl interaction with RTKs using a Cbl mutant (G306E) promotes expression of osteoblast markers (Runx2, alkaline phosphatase, type 1 collagen, osteocalcin) and increases osteogenic differentiation in clonal bone marrow-derived hMSCs and primary hMSCs. Analysis of molecular mechanisms revealed that the Cbl mutant increased PDGF receptor α and FGF receptor 2 but not EGF receptor expression in hMSCs, resulting in increased ERK1/2 and PI3K signaling. Pharmacological inhibition of FGFR or PDGFR abrogated in vitro osteogenesis induced by the Cbl mutant. The data reveal that specific inhibition of Cbl interaction with RTKs promotes the osteogenic differentiation program in hMSCs in part by decreased Cbl-mediated PDGFRα and FGFR2 ubiquitination, providing a novel mechanistic approach targeting Cbl to promote the osteogenic capacity of hMSCs.     

Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway

Physical stimuli play critical roles in the development, regeneration, and pathology of many mesenchymal tissues, most notably bone. While mature bone cells, such as osteoblasts and osteocytes, are clearly involved in these processes, the role of their progenitors in mechanically mediated tissue responses is unknown. In this study, we investigated the effect of cyclic substrate deformation on the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs). Application of equibiaxial cyclic strain (3%, 0.25Hz) to hMSCs cultured in osteogenic media inhibited proliferation and stimulated a 2.3-fold increase in matrix mineralization over unstrained cells. The strain stimulus activated the extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase pathways, but had no effect on c-Jun N-terminal kinase phosphorylation or activity. Strain-induced mineralization was largely mediated by ERK1/2 signaling, as inhibition of ERK1/2 attenuated calcium deposition by 55%. Inhibition of the p38 pathway resulted in a more mature osteogenic phenotype, suggesting an inhibitory role for p38 signaling in the modulation of strain-induced osteogenic differentiation. These results demonstrate that mechanical signals regulate hMSC function, suggesting a critical role for physical stimulation of this specific cell population in mesenchymal tissue formation.     

ERK2 protein regulates the proliferation of human mesenchymal stem cells without affecting their mobilization and differentiation potential

Human Mesenchymal Stem Cells (hMSC), derived mainly from adult bone marrow, are valuable models for the study of processes involved in stem cell self-renewal and differentiation. As the Extracellular signal-Regulated Kinase (ERK) signalling pathway is a major contributor to cellular growth, differentiation and survival, we have studied the functions of this kinase in hMSC activity. Ablation of ERK2 gene expression (but not ERK1) by RNA interference significantly reduced proliferation of hMSC. This reduction was due to a defect in Cyclin D1 expression and subsequent arrest in the G0/G1 phase of the cell cycle. hMSC growth is enhanced through culture medium supplementation with growth factors (GFs) such as Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF) or Epidermal Growth Factor (EGF). However, these supplements could not rescue the defect observed after ERK2 knockdown, suggesting a common signalling pathway used by these GFs for proliferation. In contrast, ERK1/2 may be dissociated from chemotactic signalling induced by the same GFs. Additionally, hMSCs were capable of differentiating into adipocytes even in the absence of either ERK1 or ERK2 proteins. Our data show that hMSCs do not require cell division to enter the adipogenic differentiation process, indicating that clonal amplification of these cells is not a critical step. However, cell-cell contact seems to be an essential requirement to be able to differentiate into mature adipocytes.     

Inhibition of gamma-secretases alters both proliferation and differentiation of mesenchymal stem cells

INTRODUCTION: Human mesenchymal stem cell (hMSC) proliferation and development is regulated by many signalling pathways. gamma-Secretases play an important role in Notch signalling as well as other processes that are involved in developmental decisions, but their role in hMSC proliferation and cell fate decisions has not been explored. OBJECTIVE: To investigate the role of gamma-secretases in hMSC proliferation and differentiation. MATERIALS AND METHODS: Using the gamma-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), we investigated their role in hMSC growth and differentiation to chondrogenic, osteogenic and adipogenic fates. RESULTS: We found that inhibiting gamma-secretases reduced the rate of hMSC proliferation, and altered hMSC differentiation in vitro. Addition of DAPT had an inhibitory effect on chondrogenesis resulting in impaired cartilage matrix production and altered chondrocyte morphology. DAPT treated chrodrocytic pellets had reduced levels of Hes1 and Hey1 suggesting that these effects are mediated via Notch signalling. Addition of the DAPT inhibitor to osteogenic cultures did not alter the appearance of bone markers, however, adipogenesis occurred in these cultures in a DAPT concentration-dependent manner. DAPT did not enhance adipogenesis in the presence of a potent adipogenic cocktail, but had an adipogenic effect when combined with dexamethasone only. CONCLUSION: We conclude that gamma-secretases play an important role in both hMSC proliferation and differentiation.     

P38 and ERK1/2 MAPKs Act in Opposition to Regulate BMP9-Induced Osteogenic Differentiation of Mesenchymal Progenitor Cells

Although previous studies have demonstrated that BMP9 is highly capable of inducing osteogenic differentiation and bone formation, the precise molecular mechanism involved remains to be fully elucidated. In this current study, we explore the possible involvement and detail effects of p38 and ERK1/2 MAPKs on BMP9-indcued osteogenic differentiation of mesenchymal progenitor cell (MPCs). We find that BMP9 simultaneously stimulates the activation of p38 and ERK1/2 in MPCs. BMP9-induced early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as matrix mineralization and osteocalcin (OC) are inhibited by p38 inhibitor SB203580, whereas enhanced by ERK1/2 inhibitor PD98059. BMP9-induced activation of Runx2 and Smads signaling are reduced by SB203580, and yet increased by PD98059 in MPCs. The in vitro effects of inhibitors are reproduced with adenoviruses expressing siRNA targeted p38 and ERK1/2, respectively. Using mouse calvarial organ culture and subcutaneous MPCs implantation, we find that inhibition of p38 activity leads to significant decrease in BMP9-induced osteogenic differentiation and bone formation, however, blockage of ERK1/2 results in effective increase in BMP9-indcued osteogenic differentiation in vivo. Together, our results reveal that p38 and ERK1/2 MAPKs are activated in BMP9-induced osteogenic differentiation of MPCs. What is most noteworthy, however, is that p38 and ERK1/2 act in opposition to regulate BMP9-induced osteogenic differentiation of MPCs.     

Vitamin C induces periodontal ligament progenitor cell differentiation via activation of ERK pathway mediated by PELP1

The differentiation of periodontal ligament (PDL) progenitor cells is important for maintaining the homeostasis of PDL tissue and alveolar bone. Vitamin C (VC), a water-soluble nutrient that cannot be biosynthesized by humans, is vital for mesenchymal stem cells differentiation and plays an important role in bone remodeling. Therefore, the objective of this study was to determine the function and mechanism of VC in PDL progenitor cells osteogenic differentiation at the molecular level. We demonstrated that VC could induce the osteogenic differentiation and maturation of PDL progenitor cell without other osteogenic agents. During the process, VC preferentially activated ERK1/2 but did not affect JNK or p38. Co-treatment with ERK inhibitor effectively decreased the Vitamin C-induced expression of Runx2. ERK inhibitor also abrogated Vitamin C-induced the minimized nodules formation. PELP1, a nuclear receptor co-regulator, was up-regulated under VC treatment. PELP1 knockdown inhibited ERK phosphorylation. The overexpression of PELP1 had a positive relationship with Runx2 expression. Taken together, we could make a conclude that VC induces the osteogenic differentiation of PDL progenitor cells via PELP1-ERK axis. Our fi nding implies that VC may have a potential in the regeneration medicine and application to periodontitis treatment.     

Induction of osteogenic differentiation of human mesenchymal stem cells by histone deacetylase inhibitors

Valproic acid (VPA) has been used as an anticonvulsant agent for the treatment of epilepsy, as well as a mood stabilizer for the treatment of bipolar disorder, for several decades. The mechanism of action for these effects remains to be elucidated and is most likely multifactorial. Recently, VPA has been reported to inhibit histone deacetylase (HDAC) and HDAC has been reported to play roles in differentiation of mammalian cells. In this study, the effects of HDAC inhibitors on differentiation and proliferation of human adipose tissue-derived stromal cells (hADSC) and bone marrow stromal cells (hBMSC) were determined. VPA increased osteogenic differentiation in a dose dependent manner. The pretreatment of VPA before induction of differentiation also showed stimulatory effects on osteogenic differentiation of hMSC. Trichostatin A (TSA), another HDAC inhibitor, also increased osteogenic differentiation, whereas valpromide (VPM), a structural analog of VPA which does not possess HDAC inhibitory effects, did not show any effect on osteogenic differentiation on hADSC. RT-PCR and Real-time PCR analysis revealed that VPA treatment increased osterix, osteopontin, BMP-2, and Runx2 expression. The addition of noggin inhibited VPA-induced potentiation of osteogenic differentiation. VPA inhibited proliferation of hADSC and hBMSC. Our results suggest that VPA enhance osteogenic differentiation, probably due to inhibition of HDAC, and could be useful for in vivo bone engineering using hMSC.     

The response of human mesenchymal stem cells to osteogenic signals and its impact on bone tissue engineering

Bone tissue engineering using human mesenchymal stem cells (hMSCs) is a multidisciplinary field that aims to treat patients with trauma, spinal fusion and large bone defects. Cell-based bone tissue engineering encompasses the isolation of multipotent hMSCs from the bone marrow of the patient, in vitro expansion and seeding onto porous scaffold materials. In vitro pre-differentiation of hMSCs into the osteogenic lineage augments their in vivo bone forming capacity. Differentiation of hMSCs into bone forming osteoblasts is a multi-step process regulated by various molecular signaling pathways, which warrants a thorough understanding of these signaling cues for the efficient use of hMSCs in bone tissue engineering. Recently, there has been a surge of knowledge on the molecular cues regulating osteogenic differentiation but extrapolation to hMSC differentiation is not guaranteed, because of species- and cell-type specificity. In this review, we describe a number of key osteogenic signaling pathways, which directly or indirectly regulate osteogenic differentiation of hMSCs. We will discuss how and to what extent the process is different from that in other cell types with special emphasis on applications in bone tissue engineering.