Transforming growth factor-beta1 regulates the fate of cultured spinal cord-derived neural progenitor cells

Abstract. Objectives: We have evaluated the physiological roles of transforming growth factor‐β1 (TGF‐β1) on differentiation, migration, proliferation and anti‐apoptosis characteristics of cultured spinal cord‐derived neural progenitor cells. Methods: We have used neural progenitor cells that had been isolated and cultured from mouse spinal cord tissue, and we also assessed the relevant reaction mechanisms using an activin‐like kinase (ALK)‐specific inhibitory system including an inhibitory RNA, and found that it involved potential signalling molecules such as phosphatidylinositol‐3‐OH kinase (PI3K)/Akt and mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK1/2). Results and Conclusions: Transforming growth factor‐β1‐mediated cell population growth was activated after treatment and was also effectively blocked by an ALK41517‐synthetic inhibitor (4‐(5‐benzo(1,3) dioxol‐5‐yl‐4‐pyridine‐2‐yl‐1H‐imidazole‐2‐yl) benzamide (SB431542) and ALK siRNA, thereby indicating the involvement of SMAD2 in the TGF‐β1‐mediated growth and migration of these neural progenitors cells (NPC). In the present study, TGF‐β1 actively induced NPC migration in vitro. Furthermore, TGF‐β1 demonstrated extreme anti‐apoptotic behaviour against hydrogen peroxide‐mediated apoptotic cell death. At low dosages, TGF‐β1 enhanced (by approximately 76%) cell survival against hydrogen peroxide treatment via inactivation of caspase‐3 and ‐9. TGF‐β1‐treated NPCs down‐regulated Bax expression and cytochrome c release; in addition, the cells showed up‐regulated Bcl‐2 and thioredoxin reductase 1. They also had increased p38, Akt and ERK1/2 phosphorylation, showing the involvement of both the PI3K/Akt and MAPK/ERK1/2 pathways in the neuroprotective effects of TGF‐β1. Interestingly, these effects operate on specific subtypes of cells, including neurones, neural progenitor cells and astrocytes in cultured spinal cord tissue‐derived cells. Lesion sites of spinal cord‐overexpressing TGF‐β1‐mediated prevention of cell death, cell growth and migration enhancement activity have been introduced as a possible new basis for therapeutic strategy in treatment of neurodegenerative disorders, including spinal cord injuries.     

TRIM32-dependent transcription in adult neural progenitor cells regulates neuronal differentiation

In the adult mammalian brain, neural stem cells in the subventricular zone continuously generate new neurons for the olfactory bulb. Cell fate commitment in these adult neural stem cells is regulated by cell fate-determining proteins. Here, we show that the cell fate-determinant TRIM32 is upregulated during differentiation of adult neural stem cells into olfactory bulb neurons. We further demonstrate that TRIM32 is necessary for the correct induction of neuronal differentiation in these cells. In the absence of TRIM32, neuroblasts differentiate slower and show gene expression profiles that are characteristic of immature cells. Interestingly, TRIM32 deficiency induces more neural progenitor cell proliferation and less cell death. Both effects accumulate in an overproduction of adult-generated olfactory bulb neurons of TRIM32 knockout mice. These results highlight the function of the cell fate-determinant TRIM32 for a balanced activity of the adult neurogenesis process.     

Ectopic expression of p27Kip1 in oligodendrocyte progenitor cells results in cell-cycle growth arrest

Oligodendrocyte differentiation is a complex process believed to be controlled by an intrinsic mechanism associated with cell-cycle arrest. Recently, the cell-cycle inhibitor protein p27^Kip1 has been proposed as a key element in causing growth arrest of oligodendrocyte precursor cells. To investigate the effects of p27 upon oligodendrocyte cell development, we have introduced the p27 cDNA in oligodendrocyte progenitor cells using an adenovirus vector. Progenitor cells normally express low levels of p27. After adenoviral infection and p27 overexpression, progenitor cells were able to undergo cell-cycle arrest, even in the presence of strong mitogens. The effects of p27 were shown to be directly upon cyclin-dependent kinase-2 (CDK2), the protein kinase complex responsible for G1/S transition, as immunodepletion of oligodendrocyte extracts of p27 protein resulted in the activation of CDK2 activity. However, cells that became growth arrested owing to infection with p27 adenovirus did not display conventional oligodendrocyte differentiation markers, such as O4 or O1. Taken together, these data provide mechanistic evidence indicating that p27 is primarily involved in oligodendroglial progenitor proliferation by inhibiting CDK2 activity and inducing oligodendrocyte cell-cycle arrest. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 431–440, 1998     

Epidermal growth factor regulates the development of stem and progenitor Leydig cells in rats

Epidermal growth factor (EGF) has many physiological roles. However, its effects on stem and progenitor Leydig cell development remain unclear. Rat stem and progenitor Leydig cells were cultured with different concentrations of EGF alone or in combination with EGF antagonist, erlotinib or cetuximab. EGF (1 and 10 ng/mL) stimulated the proliferation of stem Leydig cells on the surface of seminiferous tubules and isolated CD90⁺ stem Leydig cells and progenitor Leydig cells but it blocked their differentiation. EGF also exerted anti‐apoptotic effects of progenitor Leydig cells. Erlotinib and cetuximab are able to reverse EGF‐mediated action. Gene microarray and qPCR of EGF‐treated progenitor Leydig cells revealed that the down‐regulation of steroidogenesis‐related proteins (Star and Hsd3b1) and antioxidative genes. It was found that EGF acted as a proliferative agent via increasing phosphorylation of AKT1. In conclusion, EGF stimulates the proliferation of rat stem and progenitor Leydig cells but blocks their differentiation.     

Spa-1 regulates the maintenance and differentiation of human embryonic stem cells

Human embryonic stem cells (hESCs) are pluripotent, whereby they can proliferate endlessly and differentiate into many different cell types. At the molecular level, little is known of the mechanisms underlying their capability for self-renewal and differentiation. In the present study, we established two new hESC lines (AMC-hES1 and AMC-hES2) and demonstrated the existence of a regulator that may be a key molecule in hESC dynamics. Spa-1 is a principal Ras-proximate 1 (Rap1) GTPase-activating protein in hematopoietic progenitor cells that regulates Rap1-related signal transduction and is expressed restrictively in human adult tissues (bone marrow, thymus, and spleen). To investigate its functions in hESCs, we examined spa-1 expression profiles during hESC differentiation and used RNA interference (RNAi) to downregulate spa-1 in these cells. Our results show that Spa-1 is expressed in undifferentiated hESCs and is downregulated during hESC differentiation. In addition, the process of passing from the mode of self-renewal to that of differentiation in hESCs was regulated by spa-1 via Rap1/Raf/mitogen-activated protein kinase kinase/extracellular signal-related kinase signaling. An RNAi expression vector against spa-1 (pSUPER.retro.puro) was transfected into hESCs, which were seen to differentiate into three germ layers in spite of being in the undifferentiated condition. Based on our findings, therefore, it appears that spa-1 may be involved in hESC dynamics, and our results provide fundamental information regarding the self-renewal and differentiation of hESCs.     

Echovirus 1 internalization negatively regulates epidermal growth factor receptor downregulation

We have demonstrated previously that the human picornavirus Echovirus 1 (EV1) triggers an infectious internalization pathway that follows closely, but seems to stay separate, from the epidermal growth factor receptor (EGFR) pathway triggered by epidermal growth factor (EGF). Here, we confirmed by using live and confocal microscopy that EGFR and EV1 vesicles are following intimately each other but are distinct entities with different degradation kinetics. We show here that despite being sorted to different pathways and located in distinct endosomes, EV1 inhibits EGFR downregulation. Simultaneous treatment with EV1 and EGF led to an accumulation of EGFR in cytoplasmic endosomes, which was evident already 15 min p.i. and more pronounced after 2 hr p.i. EV1 treatment led to reduced downregulation, which was proven by increased total cellular amount of EGFR. Confocal microscopy studies revealed that EGFR accumulated in large endosomes, presumably macropinosomes, which were not positive for markers of the early, recycling, or late endosomes/lysosomes. Interestingly, EV1 did not have a similar blocking effect on bulk endosomal trafficking or transferrin recycling along the clathrin pathway suggesting that EV1 did not have a general effect on cellular trafficking pathways. Importantly, EGF treatment increased EV1 infection and increased cell viability during infection. Simultaneous EV1 and EGF treatment seemed to moderately enhance phosphorylation of protein kinase C α. Furthermore, similar phenotype of EGFR trafficking could be produced by phorbol 12‐myristate 13‐acetate treatment, further suggesting that activated protein kinase C α could be contributing to EGFR phenotype. These results altogether demonstrate that EV1 specifically affects EGFR trafficking, leading to EGFR downregulation, which is beneficial to EV1 infection.