β‐Catenin and Rho GTPases as downstream targets of TGF‐β1 during pulp repair

TGF‐β1 (transforming growth factor‐β1) plays a central role in regulating proliferation, migration and differentiation of dental pulp cells during the repair process after tooth injury. Our previous study showed that p38 mitogen‐activated protein kinase may act downstream of TGF‐β1 signalling to effect the differentiation of dental pulp cells. However, the molecular mechanisms that trigger and regulate the process remain to be elucidated. TGF‐β1 interacts with signalling pathways such as Wnt/β‐catenin and Rho to induce diverse biological effects. TGF‐β1 activates β‐catenin signalling, increases β‐catenin nuclear translocation and interacts with LEF/TCF to regulate gene expression. Morphologic changes in response to TGF‐β1 are associated with activation of Rho GTPases, but are abrogated by inhibitors of Rho‐associated kinase, a major downstream target of Rho. These results suggest that the Wnt/β‐catenin and Rho pathways may mediate the downstream events of TGF‐β1 signalling.     

Akt‐ and Erk‐mediated regulation of proliferation and differentiation during PDGFRβ‐induced MSC self‐renewal

Understanding the mechanisms that direct mesenchymal stem cell (MSC) self‐renewal fate decisions is a key to most tissue regenerative approaches. The aim of this study here was to investigate the mechanisms of action of platelet‐derived growth factor receptor β (PDGFRβ) signalling on MSC proliferation and differentiation. MSC were cultured and stimulated with PDGF‐BB together with inhibitors of second messenger pathways. Cell proliferation was assessed using ethynyl‐2′‐deoxyuridine and phosphorylation status of signalling molecules assessed by Western Blots. To assess differentiation potentials, cells were transferred to adipogenic or osteogenic media, and differentiation assessed by expression of differentiation association genes by qRT‐PCR, and by long‐term culture assays. Our results showed that distinct pathways with opposing actions were activated by PDGF. PI3K/Akt signalling was the main contributor to MSC proliferation in response to activation of PDGFRβ. We also demonstrate a negative feedback mechanism between PI3K/Akt and PDGFR‐β expression. In addition, PI3K/Akt downstream signal cascades, mTOR and its associated proteins p70S6K and 4E‐BP1 were involved. These pathways induced the expression of cyclin D1, cyclin D3 and CDK6 to promote cell cycle progression and MSC proliferation. In contrast, activation of Erk by PDGFRβ signalling potently inhibited the adipocytic differentiation of MSCs by blocking PPARγ and CEBPα expression. The data suggest that PDGFRβ‐induced Akt and Erk pathways regulate opposing fate decisions of proliferation and differentiation to promote MSC self‐renewal. Thus, activation of multiple intracellular cascades is required for successful and sustainable MSC self‐renewal strategies.     

Cdc6 is a rate-limiting factor for proliferative capacity during HL60 cell differentiation

The DNA replication (or origin) licensing pathway represents a critical step in cell proliferation control downstream of growth signalling pathways. Repression of origin licensing through down-regulation of the MCM licensing factors (Mcm2-7) is emerging as a ubiquitous route for lowering proliferative capacity as metazoan cells exit the cell division cycle into quiescent, terminally differentiated and senescent "out-of-cycle" states. Using the HL60 monocyte/macrophage differentiation model system and a cell-free DNA replication assay, we have undertaken direct biochemical investigations of the coupling of origin licensing to the differentiation process. Our data show that down-regulation of the MCM loading factor Cdc6 acts as a molecular switch that triggers loss of proliferative capacity during early engagement of the somatic differentiation programme. Consequently, addition of recombinant Cdc6 protein to in vitro replication reactions restores DNA replication competence in nuclei prepared from differentiating cells. Differentiating HL60 cells over-expressing either wild-type Cdc6 or a CDK phosphorylation-resistant Cdc6 mutant protein (Cdc6A4) exhibit an extended period of cell proliferation compared to mock-infected cells. Notably, differentiating HL60 cells over-expressing the Cdc6A4 mutant fail to down-regulate Cdc6 protein levels, suggesting that CDK phosphorylation of Cdc6 is linked to its down-regulation during differentiation and the concomitant decrease in cell proliferation. In this experimental model, Cdc6 therefore plays a key role in the sequential molecular events leading to repression of origin licensing and loss of proliferative capacity during execution of the differentiation programme.     

Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation.

Previously, we showed that expression of a dominant-negative form of the transforming growth factor beta (TGF-beta) type II receptor in skeletal tissue resulted in increased hypertrophic differentiation in growth plate and articular chondrocytes, suggesting a role for TGF-beta in limiting terminal differentiation in vivo. Parathyroid hormone-related peptide (PTHrP) has also been demonstrated to regulate chondrocyte differentiation in vivo. Mice with targeted deletion of the PTHrP gene demonstrate increased endochondral bone formation, and misexpression of PTHrP in cartilage results in delayed bone formation due to slowed conversion of proliferative chondrocytes into hypertrophic chondrocytes. Since the development of skeletal elements requires the coordination of signals from several sources, this report tests the hypothesis that TGF-beta and PTHrP act in a common signal cascade to regulate endochondral bone formation. Mouse embryonic metatarsal bone rudiments grown in organ culture were used to demonstrate that TGF-beta inhibits several stages of endochondral bone formation, including chondrocyte proliferation, hypertrophic differentiation, and matrix mineralization. Treatment with TGF-beta1 also stimulated the expression of PTHrP mRNA. PTHrP added to cultures inhibited hypertrophic differentiation and matrix mineralization but did not affect cell proliferation. Furthermore, terminal differentiation was not inhibited by TGF-beta in metatarsal rudiments from PTHrP-null embryos; however, growth and matrix mineralization were still inhibited. The data support the model that TGF-beta acts upstream of PTHrP to regulate the rate of hypertrophic differentiation and suggest that TGF-beta has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation.     

BDNF alters ERK/p38 MAPK activity ratios to promote differentiation in growth plate chondrocytes

The ERK and p38 MAPK pathways are well-known transducers of signals that regulate proliferation and differentiation, but precisely how these pathways control growth plate chondrocyte development is unclear. For example, the ERK pathway has been reported to be required by some investigators but inhibitory to chondrocyte development by others. Moreover, how these two pathways interact to regulate chondrocyte development is even less clear. Using primary bovine growth plate chondrocytes and murine ATDC5 cells, we demonstrate that the ERK and p38 pathways have opposing effects on proliferation but are both absolutely required for differentiation. Two factors that promote chondrocyte differentiation, brain-derived neurotrophic factor (BDNF) and C-type natriuretic peptide, increase p38 activity while decreasing, but not completely inhibiting, ERK activity. The attenuation of ERK activity by BDNF occurs via p38-dependent raf-1 inhibition. The inhibition of raf-1 by p38 is direct, because purified p38 protein inhibits the kinase activity of purified active raf-1 as well as raf-1 immunoprecipitated from chondrocyte lysates. Moreover, IGF-I, which stimulates proliferation, suppresses p38 activation. This work describes a model wherein unopposed IGF-I promotes high ERK/p38 activity ratios favoring proliferation, whereas BDNF signals a transition to differentiation by decreasing the ERK/p38 activity ratio without completely inhibiting ERK, which involves the direct inhibition of raf-1 by p38.     

TGF-β regulates β-catenin signaling and osteoblast differentiation in human mesenchymal stem cells

Human adult bone marrow-derived skeletal stem cells a.k.a mesenchymal stem cells (hMSCs) have been shown to be precursors of several different cellular lineages, including osteoblast, chondrocyte, myoblast, adipocyte, and fibroblast. Several studies have shown that cooperation between transforming growth factor β (TGF-β) and Wnt/β-catenin signaling pathways plays a role in controlling certain developmental events and diseases. Our previous data showed that agents like TGF-β, cooperation with Wnt signaling, promote chondrocyte differentiation at the expense of adipocyte differentiation in hMSCs. In this study, we tested mechanisms by which TGF-β activation of β-catenin signaling pathway and whether these pathways interact during osteoblast differentiation of hMSCs. With selective small chemical kinase inhibitors, we demonstrated that TGF-β1 requires TGF-β type I receptor ALK-5, Smad3, phosphoinositide 3-kinases (PI3K), and protein kinase A (PKA) to stabilize β-catenin, and needs ALK-5, PKA, and JNK to inhibit osteoblastogenesis in hMSCs. Knockdown of β-catenin with siRNA stimulated alkaline phosphatase activity and antagonized the inhibitory effects of TGF-β1 on bone sialoprotein (BSP) expression, suggested that TGF-β1 cooperated with β-catenin signaling in inhibitory of osteoblastogenesis in hMSCs. In summary, TGF-β1 activates β-catenin signaling pathway via ALK-5, Smad3, PKA, and PI3K pathways, and modulates osteoblastogenesis via ALK5, PKA, and JNK pathways in hMSCs; the interaction between TGF-β and β-catenin signaling supports the view that β-catenin signaling is a mediator of TGF-β's effects on osteoblast differentiation of hMSCs.     

TGF-beta/Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage

Endochondral ossification begins from the condensation and differentiation of mesenchymal cells into cartilage. The cartilage then goes through a program of cell proliferation, hypertrophic differentiation, calcification, apoptosis, and eventually is replaced by bone. Unlike most cartilage, articular cartilage is arrested before terminal hypertrophic differentiation. In this study, we showed that TGF-beta/Smad3 signals inhibit terminal hypertrophic differentiation of chondrocyte and are essential for maintaining articular cartilage. Mutant mice homozygous for a targeted disruption of Smad3 exon 8 (Smad3(ex8/ex8)) developed degenerative joint disease resembling human osteoarthritis, as characterized by progressive loss of articular cartilage, formation of large osteophytes, decreased production of proteoglycans, and abnormally increased number of type X collagen-expressing chondrocytes in synovial joints. Enhanced terminal differentiation of epiphyseal growth plate chondrocytes was also observed in mutant mice shortly after weaning. In an in vitro embryonic metatarsal rudiment culture system, we found that TGF-beta1 significantly inhibits chondrocyte differentiation of wild-type metatarsal rudiments. However, this inhibition is diminished in metatarsal bones isolated from Smad3(ex8/ex8) mice. These data suggest that TGF-beta/Smad3 signals are essential for repressing articular chondrocyte differentiation. Without these inhibition signals, chondrocytes break quiescent state and undergo abnormal terminal differentiation, ultimately leading to osteoarthritis.     

Cell responses to FGFR3 signalling: growth, differentiation and apoptosis

FGFR3 is a receptor tyrosine kinase (RTK) of the FGF receptor family, known to have a negative regulatory effect on long bone growth. Fgfr3 knockout mice display longer bones and, accordingly, most germline-activating mutations in man are associated with dwarfism. Somatically, some of the same activating mutations are associated with the human cancers multiple myeloma, cervical carcinoma and carcinoma of the bladder. How signalling through FGFR3 can lead to either chondrocyte apoptosis or cancer cell proliferation is not fully understood. Although FGFR3 can be expressed as two main splice isoforms (IIIb or IIIc), there is no apparent link with specific cell responses, which may rather be associated with the cell type or its differentiation status. Depending on cell type, differential activation of STAT proteins has been observed. STAT1 phosphorylation seems to be involved in inhibition of chondrocyte proliferation while activation of the ERK pathway inhibits chondrocyte differentiation and B-cell proliferation (as in multiple myeloma). The role of FGFR3 in epithelial cancers (bladder and cervix) is not known. Some of the cell specificity may arise via modulation of signalling by crosstalk with other signalling pathways. Recently, inhibition of the ERK pathway in achondroplastic mice has provided hope for an approach to the treatment of dwarfism. Further understanding of the ability of FGFR3 to trigger different responses depending on cell type and cellular context may lead to treatments for both skeletal dysplasias and cancer.     

Wnt5b regulates mesenchymal cell aggregation and chondrocyte differentiation through the planar cell polarity pathway

Although genetic evidence has demonstrated a role for Wnt5b during cartilage and limb development, little is known about the mechanisms underlying Wnt5b-regulated chondrocyte differentiation. We observed that Wnt5b inhibited chondrocyte hypertrophy and expression of type X collagen. In addition, Wnt5b regulated the overall size of chondrogenic cultures, suggesting that Wnt5b regulates other processes involved in cartilage development. We therefore investigated the signaling pathways by which Wnt5b influences differentiation. Wnt5b activated known calcium-dependent signaling pathways and JNK, a component of the planar cell polarity pathway. Since the planar cell polarity pathway regulates process such as cell migration and cell aggregation that are involved in limb development, we assayed for effects of Wnt5b on these processes. We observed a marked increase chondroprogenitor cell migration with Wnt5b expression. This effect was blocked by inhibition of JNK, but not by inhibition of other Wnt5b-responsive factors. Expression of Wnt5b also disrupted the cellular aggregation associated with mesenchymal condensation. Decreased aggregation was associated with reduced cadherin expression as well as increased cadherin receptor turnover. This increase in cadherin receptor turnover was associated with an increase in Src-dependent beta-catenin phosphorylation downstream of Wnt5b. Our data demonstrate that not only does Wnt5b inhibit chondrocyte hypertrophy, but document a novel role for Wnt5b in modulating cellular migration through the JNK-dependent and cell adhesion through an activation of Src and subsequent cadherin receptor turnover.     

PKCε acts as negative allosteric modulator of EGF receptor signalling

Protein kinase C ε (PKCε) is a transforming oncogene and plays a pivotal role in numerous cellular processes including proliferation, invasion and differentiation. Recently, we described a function of PKCε as a scaffold protein linking PLCγ1 to the EGFR module. Here, in the head and neck squamous carcinoma cell line (HNSCC) FaDu we demonstrate that over-expressed PKCε may be associated with the EGFR. This is linked with the consecutive inhibition of the recruitment of PLCγ1 to the EGFR, of the catalytical activation of PLCγ1 by EGF, and of the PLCγ1-mediated effect of EGF on cell proliferation. These effects are independent of the catalytical as well as the scaffold activity of PKCε but are a function of the cellular expression level of PKCε. In contrast to FaDu cells where the PLCγ1 pathway was selectively affected, in three other HNSCC cell lines investigated over-expression of PKCε resulted in association with EGFR and, subsequently, in either partial (ERK and Akt or PLCγ1 and Akt) or complete (ERK, PLCγ1 and Akt) inhibition of the main EGFR signalling pathways. Together, our data suggest that in particular carcinoma cells highly expressed PKCε may act as negative allosteric modulator of EGFR signalling. This novel function of PKCε provides also the first indication that the EGFR may be a target for allosteric modulation by accessory proteins.     

Stathmin inhibits proliferation and differentiation of dental pulp stem cells via sonic hedgehog/Gli

The mineralization of dental pulp stem cells is an important factor in the tissue engineering of teeth, but the mechanism is not yet obvious. This study aimed to identify the effect of Stathmin on the proliferation and osteogenic/odontoblastic differentiation of human dental pulp stem cells (hDPSCs) and to explore whether the Shh signalling pathway was involved in this regulation. First, Stathmin was expressed in the cytoplasm and on the cell membranes of hDPSCs by cell immunofluorescence. Then, by constructing a lentiviral vector, the expression of Stathmin in hDPSCs was inhibited. Treatment with Stathmin shRNA (shRNA‐Stathmin group) inhibited the ability of hDPSCs to proliferate, as demonstrated by a CCK8 assay and flow cytometry analysis, and suppressed the osteogenic/odontoblastic differentiation ability, as demonstrated by alizarin red S staining and osteogenic/odontoblastic differentiation‐related gene (ALP, BSP, OCN, DSPP) activity, compared to that of hDPSCs from the control shRNA group. Molecular analyses showed that the Shh/GLI1 signalling pathway was inhibited when Stathmin was silenced, and purmorphamine, the Shh signalling pathway activator, was added to hDPSCs in the shRNA‐Stathmin group, real‐time PCR and Western blotting confirmed that expression of Shh and its downstream signalling molecules PTCH1, SMO and GLI1 increased significantly. After activating the Shh signalling pathway, the proliferation of hDPSCs increased markedly, as demonstrated by a CCK8 assay and flow cytometry analysis; osteogenic/odontoblastic differentiation‐related gene (ALP, BSP, OCN, DSPP) expression also increased significantly. Collectively, these findings firstly revealed that Stathmin‐Shh/GLI1 signalling pathway plays a positive role in hDPSC proliferation and osteogenic/odontoblastic differentiation.     

l-Cysteine-induced up-regulation of the low-density lipoprotein receptor is mediated via a transforming growth factor-alpha signalling pathway

Sulphur-containing amino acids regulate plasma cholesterol levels in animals and humans. However, their mechanism of action remains unclear. Low-density lipoprotein receptor (LDLR) plays an important role in cholesterol metabolism. We therefore investigated the effects of sulphur-containing amino acids on the expression of LDLR in hepatocytes. HepG2 cells were cultured in Dulbecco’s Modified Eagle’s Medium with or without sulphur-containing amino acids and cysteine-containing compounds. We found that l-cysteine increased LDLR mRNA and enhanced LDLR gene promoter activity through the extracellular-signal-related kinase and p38 mitogen-activated protein kinase signalling pathways in HepG2 cells. Moreover, we observed that l-cysteine stimulated the release of transforming growth factor-alpha (TGF-α) and that TGF-α increased the LDLR mRNA levels. This study provides a report of the l-cysteine mediated up-regulation of the LDLR expression via TGF-α signalling pathway. Our findings provide insights into cholesterol homeostasis and amino acid signalling.     

Amino acid signalling and the integration of metabolism

It has become clear in recent years that amino acids are not only important as substrates for various metabolic pathways but that they can also activate a nutrient-sensitive, mTOR-mediated, signalling pathway in synergy with insulin. Leucine is the most effective amino acid in this regard. The signalling pathway is antagonised by AMP-activated protein kinase. Amino acid signalling stimulates protein synthesis and inhibits (autophagic) proteolysis. In addition, many amino acids cause an increase in cell volume. Cell swelling per se stimulates synthesis of protein, glycogen, and lipid, in part by further stimulating signalling and in part by unrelated mechanisms. Amino acids also stimulate signalling in beta-cells and stimulate beta-cell growth and proliferation. This results in increased production of insulin, which enhances the anabolic (and anti-catabolic) properties of amino acids. Finally, amino acid-dependent signalling controls the production of leptin by adipocytes, and thus contributes to the regulation of appetite.     

The Increase in Retinal Cells Proliferation Induced by FGF2 is Mediated by Tyrosine and PI3 Kinases

Since 1973, multiple effects of basic fibroblast growth factor have been described in a large number of cells. These effects include proliferation, survival and differentiation. The aim of this work was to study the intracellular pathways involved in the basic fibroblast growth factor (FGF2) effect on rat retinal cells proliferation in vitro. Our data show that treatment with FGF2 increases proliferation in a concentration- and time-dependent manner. The effect of 25 ng/ml FGF2 was blocked by 10 μM genistein, a tyrosine kinase inhibitor and by 25 μM LY294002, a PI3 kinase inhibitor. The concomitant treatment with 0.3 μM chelerythrine chloride, a protein kinase C inhibitor, and 6.25 μM LY294002 also inhibited the effect of FGF2. Our results suggest that the proliferative effect of FGF2 on retinal cell cultures involves the activation of distinct kinases.     

Mechanisms of hepatocyte growth factor-mediated signaling in differentiation of pancreatic ductal epithelial cells into insulin-producing cells

Pancreatic ductal epithelial cells (PDECs) were induced to differentiate into insulin-producing cells by hepatocyte growth factor (HGF) in our previous study, but the mechanism through which this induction occurs is still unknown. HGF is a ligand that activates a tyrosine kinase encoded by the c-Met proto-oncogene. This activation is followed by indirect activation of multiple downstream signal transduction pathways (including MAPKs and the PI3K/AKT signaling pathways) that initiate various biological effects. Therefore, we speculated that the differentiation of PDECs is through either the MAPK signaling pathway or the PI3K/AKT signaling pathway. To test this hypothesis, isolated PDECs from adult rats were stimulated by adding HGF to their medium for 28 days. Then, the expression levels of several protein kinases, including MAPKs (ERK1/2, p38, and JNK) and AKT, were determined by Western blotting to determine if specific protein kinases are activated in these pathways. Subsequently, re-isolated from adult rats and cultured PDECs were pre-treated with specific inhibitors of proteins shown to be activated in these signaling pathways; these cells were then induced to differentiate by the addition of HGF. The expression levels of protein kinases were determined by Western blotting, and the differentiation rate of insulin-positive cells was determined by flow cytometry. The change of PDEC differentiation rates were compared between the groups in which cells with or without inhibitors pretreatment to determine the specific signaling pathway(s) that may be involved in HGF-induced differentiation of PDECs. After isolating PDECs and stimulating them with HGF for 28 days, the expression levels of phosphorylated ERK1/2 as well as total and phosphorylated AKT of cultured cells were significantly increased compared to the normal control group (p < 0.05), suggesting that the signaling pathways involving ERK1/2 and Akt (MEK-ERK and PI3K-AKT) are activated during HGF-induced PDEC differentiation. MEK1/2 or PI3K inhibitors were separately added to the culture medium of PDECs pre-treated with HGF. These results show that compared to the HGF-treated group, the differentiation rate of insulin-positive cells was significantly decreased in the HGF/LY294002 (PI3K inhibitor) group (13.47 ± 1.57% vs. 33.47 ± 1.34%, p < 0.05); however, the differentiation rate of insulin-positive cells was not significantly different in the HGF/PD98059 (MEK1/2 inhibitor) group. These data suggest that HGF induces PDECs to differentiate into insulin-producing cells through the PI3K/AKT signaling pathway.