[Promoting effects and mechanisms of prostaglandin E1 on proliferation and migration of endothelial cells].

OBJECTIVE: To investigate the mechanism(s) that prostaglandin E1 (PGE1) promotes human umbilical vein endothelial cell (HUVEC)proliferation and migration. METHODS: Western blot, enzyme linked immunosorbent assay, cell proliferation and cell migration tests, and tube formation were used for analyzing the roles and mechanisms of PGE1 on HUVEC; Western blot was used for analyzing the effects of PGE1 on the expression of vascular endothelial growth factor (VEGF) in rat aortic vascular smooth muscle cells (VSMC). RESULTS: PGE1 significantly increased VEGF expression of HUVEC in time and a dose dependent manner with concomitantly increased HUVEC proliferation; treatment of HUVEC with Bevacizumab apparently suppressed PGE1-stimulated VEGF expression, which led to decreased tube formation, reduced cell proliferation and migration by 41% and 38%, respectively, compared with PGE1 treatment alone; PGE1 time-dependently induced both phosphorylation of ERK and p38 in HUVEC, whereas ERK inhibitor, PD98059, or p38 inhibitor, SB203580, blocked PGE1-induced VEGF expression of HUVEC, resulting in dramatically suppression of HUVEC proliferation and migration compared with PGE1 treatment alone (60% and 55% by PD98059, 62% and 51% by SB203580, respectively); in addition, cAMP-dependent protein kinase A inhibitor, H89 or Rp-cAMP blocked PGE1-induced VEGF expression in VSMC. CONCLUSION: PGE1 promotion of proliferation, migration and tube formation of HUVEC via VEGF further provides a novel theoretical support in efficacy of PGE1 treatment of critical limb ischemia and other related diseases.

Both polymorphic variable number of tandem repeats and autoimmune regulator modulate differential expression of insulin in human thymic epithelial cells.

OBJECTIVE: Polymorphic INS-VNTR plays an important role in regulating insulin transcript expression in the human thymus that leads to either insulin autoimmunity or tolerance. The molecular mechanisms underlying the INS-VNTR haplotype-dependent insulin expression are still unclear. In this study, we determined the mechanistic components underlying the differential insulin gene expression in human thymic epithelial cells, which should have profound effects on the insulin autoimmune tolerance induction. RESEARCH DESIGN AND METHODS: A repetitive DNA region designated as a variable number of tandem repeats (VNTR) is located upstream of the human insulin gene and correlates with the incidence of type 1 diabetes. We generated six class I and two class III VNTR constructs linked to the human insulin basal promoter or SV40 heterologous promoter/enhancer and demonstrated that AIRE protein modulates the insulin promoter activities differentially through binding to the VNTR region. RESULTS: Here we show that in the presence of the autoimmune regulator (AIRE), the class III VNTR haplotype is responsible for an average of three-fold higher insulin expression than class I VNTR in thymic epithelial cells. In a protein-DNA pull-down experiment, AIRE protein is capable of binding to VNTR class I and III probes. Further, the transcriptional activation of the INS-VNTR by AIRE requires the insulin basal promoter. The VNTR sequence loses its activation activity when linked to a heterologous promoter and/or enhancer. CONCLUSIONS: These findings demonstrate a type 1 diabetes predisposition encoded by the INS-VNTR locus and a critical function played by AIRE, which constitute a dual control mechanisms regulating quantitative expression of insulin in human thymic epithelial cells.

Direct control of neurogenesis by selector factors in the fly eye: regulation of atonal by Ey and So.

During eye development, the selector factors of the Eyeless/Pax6 or Retinal Determination (RD) network control specification of organ-type whereas the bHLH-type proneural factor Atonal drives neurogenesis. Although significant progress has been made in dissecting the acquisition of ;eye identity' at the transcriptional level, the molecular mechanisms underlying the progression from neuronal progenitor to differentiating neuron remain unclear. A recently proposed model for the integration of organ specification and neurogenesis hypothesizes that atonal expression in the eye is RD-network-independent and that Eyeless works in parallel or downstream of atonal to modify the neurogenetic program. We show here that distinct cis-regulatory elements control atonal expression specifically in the eye and that the RD factors Eyeless and Sine oculis function as direct regulators. We find that these transcription factors interact in vitro and provide indirect evidence that this interaction may be required in vivo. The subordination of neurogenesis to the RD pathway in the eye provides a direct mechanism for the coordination of neurogenesis and tissue specification during sensory organ formation.

Partner specificity is essential for proper function of the SIX-type homeodomain proteins Sine oculis and Optix during fly eye development.

The development of the Drosophila visual system utilizes two members of the highly conserved Six-Homeobox family of transcription factor, Sine oculis and Optix. Although in vitro studies have detected differences in DNA-binding and interactions with some co-factors, questions remain as to what extent the activity for these two transcriptional regulators is redundant or specific in vivo. In this work, we show that the SoD mutation within the Six domain does not abolish DNA-protein interactions, but alters co-factor binding specificity to resemble that of Optix. A mutation in the same region of Optix alters its activity in vivo. We propose that the dominant mutant phenotype is primarily due to an alteration in binding properties of the Sine oculis protein and that distinct partner interactions is one important mechanism in determining significant functional differences between these highly conserved factors during eye development.

Immunization with myelin or recombinant Nogo-66/MAG in alum promotes axon regeneration and sprouting after corticospinal tract lesions in the spinal cord.

We have shown previously that immunization with myelin in incomplete Freund's adjuvant (IFA) is able to promote robust regeneration of corticospinal tract fibers in adult mice. In the present study the effectiveness of such immunization with myelin was compared to that of a combination of two axon growth inhibitors in myelin, Nogo-66 (the 66-amino-acid inhibitory region of Nogo-A) and myelin-associated glycoprotein (MAG). The effectiveness of two adjuvants, IFA and aluminum hydroxide (Alum), was also compared, the latter being one that can be used in humans. In addition, larger dorsal overhemisections were made at the lower thoracic level, which resulted in a larger scar. These studies were carried out in SJL/J mice, a mouse strain that is susceptible to autoimmune experimental allergic encephalomyelitis (EAE). None of the immunized mice developed EAE. Long-distance axon regeneration and sprouting of the corticospinal tract was seen in myelin and Nogo-66/MAG immunized mice. Alum was as effective or better than IFA as the adjuvant. Overall, the robustness of axon growth and sprouting was greater in mice immunized with myelin. The abundance of this growth was less than in our earlier work in which smaller lesions were made, pointing to the possible influence of inhibitors in the scar. This work shows, however, that axon growth inhibitors in myelin can be selectively blocked using this immunization approach to promote long-distance axon regeneration in the spinal cord.