Betacellulin regulates the proliferation and differentiation of retinal progenitor cells in vitro

Retinal progenitor cells (RPCs) hold great potential for the treatment of retinal degenerative diseases. However, their proliferation capacity and differentiation potential towards specific retinal neurons are limited, which limit their future clinical applications. Thus, it is important to improve the RPCs’ ability to proliferate and differentiate. Currently, epidermal growth factor (EGF) is commonly used to stimulate RPC growth in vitro. In this study, we find that betacellulin (BTC), a member of the EGF family, plays important roles in the proliferation and differentiation of RPCs. Our results showed that BTC can significantly promote the proliferation of RPCs more efficiently than EGF. EGF stimulated RPC proliferation through the EGFR/ErbB2‐Erk pathway, while BTC stimulated RPC proliferation more powerfully through the EGFR/ErbB2/ErbB4‐Akt/Erk pathway. Meanwhile, under differentiated conditions, the BTC‐pre‐treated RPCs were preferentially differentiated into retinal neurons, including photoreceptors, one of the most important types of cells for retinal cell replacement therapy, compared to the EGF‐pre‐treated RPCs. In addition, knockdown of endogenous BTC expression can also obviously promote RPC differentiation into retinal neuronal cells. This data demonstrate that BTC plays important roles in promoting RPC proliferation and differentiation into retinal neurons. This study may provide new insights into the study of RPC proliferation and differentiation and make a step towards the application of RPCs in the treatment of retinal degenerative diseases.     

Decellularized matrix of adipose-derived mesenchymal stromal cells enhanced retinal progenitor cell proliferation via the Akt/Erk pathway and neuronal differentiation

Background aims

Retinal progenitor cells (RPCs) are a promising cell therapy treatment for retinal degenerative diseases. However, problems with limited proliferation ability and differentiation preference toward glia rather than neurons restrict the clinical application of these RPCs. The extracellular matrix (ECM) has been recognized to provide an appropriate microenvironment to support stem cell adhesion and direct cell behaviors, such as self-renewal and differentiation.

PI3 kinase/Akt activation mediates estrogen and IGF-1 nigral DA neuronal neuroprotection against a unilateral rat model of Parkinson's disease

Recently, using the medial forebrain bundle (MFB) 6-hydroxydopmaine (6-OHDA) lesion rat model of Parkinson's disease (PD), we have demonstrated that blockade of central IGF-1 receptors (IGF-1R) attenuated estrogen neuroprotection of substantia nigra pars compacta (SNpc) DA neurons, but exacerbated 6-OHDA lesions in IGF-1 only treated rats (Quesada and Micevych [2004]: J Neurosci Res 75:107-116). This suggested that the IGF-1 system is a central mechanism through which estrogen acts to protect the nigrostriatal DA system. Moreover, these results also suggest that IGF-1R-induced intracellular signaling pathways are involved in the estrogen mechanism that promotes neuronal survival. In vitro, two convergent intracellular signaling pathways used by estrogen and IGF-1, the mitogen-activated protein kinase (MAPK/ERK), and phosphatidyl-inositol-3-kinase/Akt (PI3K/Akt), have been demonstrated to be neuroprotective. Continuous central infusions of MAPK/ERK and PI3K/Akt inhibitors were used to test the hypothesis that one or both of these signal transduction pathways mediates estrogen and/or IGF-1 neuroprotection of SNpc DA neurons after a unilateral administration of 6-OHDA into the MFB of rats. Motor behavior tests and tyrosine hydroxylase immunoreactivity revealed that the inhibitor of the PI3K/Akt pathway (LY294002) blocked the survival effects of both estrogen and IGF-1, while an inhibitor of the MAPK/ERK signaling (PD98059) was ineffective. Western blot analyses showed that estrogen and IGF-1 treatments increased PI3K/Akt activation in the SN; however, MAPK/ERK activation was decreased in the SN. Indeed, continuous infusions of inhibitors blocked phosphorylation of PI3K/Akt and MAPK/ERK. These findings indicate that estrogen and IGF-1-mediated SNpc DA neuronal protection is dependent on PI3K/Akt signaling, but not on the MAPK/ERK pathway.     

Effects of insulin and insulin-like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin-like growth factor 1 (IGF-1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF-1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF-1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF-1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF-1 promote osteoblast differentiation and mineralization in vitro, IGF-1, but not insulin, can induce osteoblast proliferation. The IGF-1-induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF-1-mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3-kinase inhibitor. Osteocalcin, an osteoblast-specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose- and time-dependent manner after insulin treatment, whereas it was decreased with IGF-1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF-1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis.     

IGF-1-Mediated Survival from Induced Death of Human Primary Cultured Retinal Pigment Epithelial Cells Is Mediated by an Akt-Dependent Signaling Pathway

Degeneration of the human retinal pigmented epithelium (hRPE) is involved in several eye disorders such as age-related macular degeneration (AMD). In this study, we investigated the protective effect of IGF-1 on human primary cultured RPE cells and its underlying mechanism. IGF-1 dose- and time-dependently promoted the survival of RPE cells from serum deprivation. Western blot showed that IGF-1 stimulated the activation of the PI3K/Akt and MAPK pathways in hRPE. Inhibition of the PI3K/Akt pathway by the PI3K-specific inhibitor, LY294002 or inhibition of Akt by Akt-specific inhibitors Akt inhibitor VIII or SN-38, or downregulation Akt with siRNA specific for Akt blocked the effect of IGF-1 on hRPE. In contrast, blockade of the MAPK pathway with a specific inhibitor PD98059 had no effect. Interestingly, vitreous IGF-1 injection reversed the inhibitory effect of light exposure (a dry AMD model) on both a wave and b wave. Immunocytochemistry showed that vitreous IGF-1 injections promoted the survival of RPE cells in rat retina and the expression of RPE65 in RPE cells from light injury. These results indicate that IGF-1 is able to protect hRPE cell from different insults in vivo and in vitro. Further detailed studies may lead the way to a therapeutic intervention for retinal diseases in which cell death is an underlying contributory mechanism.     

IGF-1 activates hEAG K(+) channels through an Akt-dependent signaling pathway in breast cancer cells: role in cell proliferation

Previous work from our laboratory has shown that human ether à go-go (hEAG) K(+) channels are crucial for breast cancer cell proliferation and cell cycle progression. In this study, we investigated the regulation of hEAG channels by an insulin-like growth factor-1 (IGF-1), which is known to stimulate cell proliferation. Acute applications of IGF-1 increased K(+) current-density and hyperpolarized MCF-7 cells. The effects of IGF-1 were inhibited by hEAG inhibitors. Moreover, IGF-1 increased mRNA expression of hEAG in a time-dependent manner in parallel with an enhancement of cell proliferation. The MCF-7 cell proliferation induced by IGF-1 is inhibited pharmacologically by Astemizole or Quinidine or more specifically using siRNA against hEAG channel. Either mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K) are known to mediate IGF-1 cell proliferative signals through the activation of extracellular signal-regulated kinase 1/2 (Erk 1/2) and Akt, respectively. In MCF-7 cells, IGF-1 rapidly stimulated Akt phosphorylation, whereas IGF-1 had little stimulating effect on Erk 1/2 which seems to be constitutively activated. The application of wortmannin was found to block the effects of IGF-1 on K(+) current. Moreover, the inhibition of Akt phosphorylation by the application of wortmannin or by a specific reduction of Akt kinase activity reduced the hEAG mRNA levels. Taken together, our results show, for the first time, that IGF-1 increases both the activity and the expression of hEAG channels through an Akt-dependent pathway. Since a hEAG channel is necessary for cell proliferation, its regulation by IGF-1 may thus play an important role in IGF-1 signaling to promote a mitogenic effect in breast cancer cells.     

Comparative signaling pathways of insulin-like growth factor-1 and brain-derived neurotrophic factor in hippocampal neurons and the role of the PI3 kinase pathway in cell survival

Insulin-like growth factor-1 (IGF-1) and brain-derived neurotrophic factor (BDNF) are trophic factors required for the viability and normal functions of various neuronal cells. However, the detailed intracellular mechanism(s) involved in these effects in neuronal cells remains to be fully elucidated. In present study, the respective intracellular signaling pathway induced by IGF-1 and BDNF and their possible role in neuronal survival were investigated. Both IGF-1 and BDNF protected hippocampal neurons from serum deprivation-induced death with IGF-1 apparently being more potent. Western blot analyses showed that both IGF-1 and BDNF induced the activation of the phosphatidylinositide 3 kinase (PI3)/Akt (protein kinase B) kinase and the mitogen-activated protein kinase (MAPK) pathways. The phosphorylation of Akt and its downstream target, FKHRL1, induced by IGF-1 was rapid and sustained while that of MAPK was transient. The reverse situation was observed for BDNF. Moreover, IGF-1 potently induced the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and its association with PI3 kinase while BDNF was weak in these assays. In contrast, the tyrosine phosphorylation of Shc proteins was dramatically stimulated by BDNF, with IGF-1 having only a minimal effect. Most interestingly, only the inhibitor of the PI3K/Akt pathway, LY294002, was able to block the survival effects of both IGF-1 and BDNF; an inhibitor of the MAPK pathway inhibitor, PD98059, being ineffective. Taken together, these data reveal that the survival properties of both IGF-1 and BDNF against serum deprivation are mediated by the activation of the PI3K/Akt, but not the MAPK, pathway in hippocampal neurons.     

Mouse retinal progenitor cell (RPC) cocultivation with retinal pigment epithelial cell culture affects features of RPC differentiation

We provide evidence that coculturing of retinal progenitor cells (RPC) with retinal pigment epithelial cells significantly biases the standard in vitro RPC differentiation patterns. In particular, in cocultivation experiments RPCs lost the ability to differentiate spontaneously and displayed approximately 2.1-2.4-fold increase in immunoreactivity to the neural stem cell marker nestin and approximately 1.6-1.7-fold increase in rod photoreceptor cell rhodopsin marker immunoreactivity. The data suggest the influence of the intercellular interaction networks on RPC differentiation.     

Microfluidic Generated EGF-Gradients Induce Chemokinesis of Transplantable Retinal Progenitor Cells via the JAK/STAT and PI3Kinase Signaling Pathways

A growing number of studies are evaluating retinal progenitor cell (RPC) transplantation as an approach to repair retinal degeneration and restore visual function. To advance cell-replacement strategies for a practical retinal therapy, it is important to define the molecular and biochemical mechanisms guiding RPC motility. We have analyzed RPC expression of the epidermal growth factor receptor (EGFR) and evaluated whether exposure to epidermal growth factor (EGF) can coordinate motogenic activity in vitro. Using Boyden chamber analysis as an initial high-throughput screen, we determined that RPC motility was optimally stimulated by EGF concentrations in the range of 20-400ng/ml, with decreased stimulation at higher concentrations, suggesting concentration-dependence of EGF-induced motility. Using bioinformatics analysis of the EGF ligand in a retina-specific gene network pathway, we predicted a chemotactic function for EGF involving the MAPK and JAK-STAT intracellular signaling pathways. Based on targeted inhibition studies, we show that ligand binding, phosphorylation of EGFR and activation of the intracellular STAT3 and PI3kinase signaling pathways are necessary to drive RPC motility. Using engineered microfluidic devices to generate quantifiable steady-state gradients of EGF coupled with live-cell tracking, we analyzed the dynamics of individual RPC motility. Microfluidic analysis, including center of mass and maximum accumulated distance, revealed that EGF induced motility is chemokinetic with optimal activity observed in response to low concentration gradients. Our combined results show that EGFR expressing RPCs exhibit enhanced chemokinetic motility in the presence of low nanomole levels of EGF. These findings may serve to inform further studies evaluating the extent to which EGFR activity, in response to endogenous ligand, drives motility and migration of RPCs in retinal transplantation paradigms.     

Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways

Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.     

Differential signalling pathways for EGF versus PDGF activation of Erk1/2 MAP kinase and cell proliferation in brown pre-adipocytes

Stimulation by both adrenergic and non-adrenergic pathways can induce proliferation of brown pre-adipocytes. To understand the signalling pathways involved in non-adrenergic stimulation of cell proliferation, we examined Erk1/2 activation. In primary cultures of mouse brown pre-adipocytes, both EGF (epidermal growth factor) and PDGF (platelet-derived growth factor) induced Erk1/2 activation. EGF-stimulated Erk1/2 activation involved Src tyrosine kinases, but not PKC or PI3K, whereas in PDGF-induced Erk1/2 activation, PI3K, PKC (probably the atypical ζ isoform) and Src were involved sequentially. Both EGF and PDGF induced PI3K-dependent Akt activation that was not involved in Erk1/2 activation. By comparing effects of signalling inhibitors (wortmannin, SH-6, TPA, Gö6983, PP2, PD98059) on EGF- and PDGF-induced Erk1/2 activation and cell proliferation (³H-thymidine incorporation), we conclude that while the signal transduction pathways initiated by these growth factors are clearly markedly different, their effects on cell proliferation can be fully explained through their stimulation of Erk1/2 activation; thus Erk1/2 is a common, essential step for stimulation of proliferation in these cells.     

Evidence for an evolutionary conserved role of homothorax/Meis1/2 during vertebrate retina development

During eye development in D. melanogaster, the TALE-homeodomain protein Homothorax (Hth) is expressed by progenitor cells ahead of the neurogenic wave front, promotes rapid proliferation of these cells and is downregulated before cells exit the cell cycle and differentiate. Here, we present evidence that hth function is partially conserved in vertebrates. Retinal progenitor cells (RPCs) in chicks and mice express two Hth-related proteins, Meis1 and Meis2 (Mrg1), in species-specific temporal sequences. Meis1 marks RPCs throughout the period of neurogenesis in the retina, whereas Meis2 is specific for RPCs prior to the onset of retinal differentiation. Transfection of Meis-inactivating constructs impaired RPC proliferation and led to microphthalmia. RNA-interference-mediated knock-down of expression indicated that progenitor cells expressing Meis1 together with Meis2 proliferate more rapidly than cells expressing Meis1 alone. Transfection of Meis-inactivating constructs reduced the expression of cyclin D1 (Ccnd1) in the eye primordium and co-transfection of cyclin D1 partially rescued RPC proliferation. Collectively, these results suggest that (1) Meis1 and Meis2, similar to hth, maintain retinal progenitor cells in a rapidly proliferating state; (2) they control the expression of some ocular-determination genes and components of the cell cycle machinery; and (3) together with the species-specific differences in Meis1/Meis2 expression, combinatorial expression of Meis family proteins might be a candidate mechanism for the differential regulation of eye growth among vertebrate species.     

Anti-apoptotic effect of retinoic acid on retinal progenitor cells mediated by a protein kinase A-dependent mechanism

Retinal progenitor cells （RPCs） are neural stem cells able to differentiate into any normal adult retinal cell type, except for pigment epithelial cells. Retinoic acid （RA） is a powerful growth/differentiation factor that generally causes growth inhibition, differentiation and/or apoptosis. In this study, we demonstrate that RA not only affects mouse RPC differentiation but also improves cell survival by reducing spontaneous apoptotic rate without affecting RPC proliferation. The enhanced cell survival was accompanied by a significant upregulation of the expression of protein kinase A （PKA） and several protein kinase C （PKC） isoforms. Treatment of cells grown in RA-free media with 8-bromoadenosine3＇,5＇-cyclic monophosphate, a known activator of PKA, resulted in an anti-apoptotic effect similar to that caused by RA; whereas the PKA inhibitor N-[2-（p-bromocinnamylamino）ethyl]-5-isoquinolinesul- fonamide dihydrochloride led to a significant （-32%） increase in apoptosis. In contrast, treatment of RPCs with any of two PKC selective inhibitors, 2,2＇,3,3＇,4,4＇-hexahydroxy-1,1 ＇-biphenyl-6,6＇-dimethanol dimethyl ether and bisindolylmaleimide XI, led to diminished apoptosis; while a PKC activator, phorbol 12-myristate 13-acetate, increased apoptosis. These and other data suggest that the effect of RA on RPC survival is mostly due to the increased anti-apoptotic activity elicited by PKA, which might in turn be antagonized by PKC. Such a mechanism is a new example of tight regulation of important biological processes triggered by RA. Although the detailed mechanisms remain to be elucidated, we provide evidence that the pro-survival effect of RA on RPCs is not mediated by changed expression of p53 or bcl-2, and appears to be independent of 15-amyloid, Fas ligand, TNF-α, ganglioside GM1 and ceramide C 16-induced apoptotic pathways.     

Convergent proliferative response and divergent morphogenic pathways induced by epicardial and endocardial signaling in fetal heart development

Heart development requires cardiomyocyte proliferation, coupled with morphogenic differentiation of the inner trabecular myocardium and the outer compact zone myocardium. In mouse embryos lacking the retinoic acid receptor RXRalpha, proliferation and morphogenesis of the compact zone fails. We demonstrated previously that epicardial cells, in response to retinoic acid, secrete an activity that promotes cell proliferation. In this study, we have investigated downstream signaling pathways that are elicited in response to this factor. We find that cells treated in culture activate PI3 kinase and Erk pathways, and that these are required for a proliferative response. In vivo, phosphorylation of Akt and GSK3beta (PI3K pathway) and of Erk1/2 and p90rsk (Erk pathway) is substantially reduced in RXRalpha-deficient heart tissue. Neuregulin, a mitogen secreted from the endocardium which promotes cardiomyocyte proliferation and trabecular differentiation, also activates proliferation via PI3K and Erk pathways. However, the epicardial factor is not neuregulin, and does not function via the neuregulin receptor. Gene markers known to be selectively expressed in trabecular or compact myocardium in vivo are differentially activated in cell culture by treatment with neuregulin or epicardial factor, and are misexpressed in RXRalpha(-/-) heart tissue. We therefore conclude that epicardial and endocardial signals converge on common proliferative components, but diverge in downstream pathways that lead to compact vs. trabecular morphogenic differentiation.     

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.