Inhibition of the Nuclear Import of Cubitus Interruptus by Roadkill in the Presence of Strong Hedgehog Signal

Hedgehog (Hh) signalling plays an important role in various developmental processes by activating the Cubitus interruptus (Ci)/Glioblastoma (Gli) family of transcription factors. In the process of proper pattern formation, Ci activity is regulated by multiple mechanisms, including processing, trafficking, and degradation. However, it remains elusive how Ci distinctly recognizes the strong and moderate Hh signals. Roadkill (Rdx) induces Ci degradation in the anterior region of the Drosophila wing disc. Here, we report that Rdx inhibited Ci activity by two different mechanisms. In the region abutting the anterior/posterior boundary, which receives strong Hh signal, Rdx inhibited the nuclear import of Ci by releasing importin α3 from Ci. In this region, Rdx negatively regulated the expression of transcription factor Knot/Collier. In farther anterior regions receiving moderate levels of Hh signal, Rdx induced Ci degradation, as reported previously. Thus, two different mechanisms by which Rdx negatively regulates Ci may play an important role in the fine-tuning of Hh responses

Drosophila Activating Transcription Factor-2 Is Involved in Stress Response via Activation by p38, but Not c-Jun NH(2)-Terminal Kinase

Activating transcription factor (ATF)-2 is a member of the ATF/cAMP response element-binding protein family of transcription factors, and its trans-activating capacity is enhanced by stress-activated protein kinases such as c-Jun NH(2)-terminal kinase (JNK) and p38. However, little is known about the in vivo roles played by ATF-2. Here, we identified the Drosophila homologue of ATF-2 (dATF-2) consisting of 381 amino acids. In response to UV irradiation and osmotic stress, Drosophila p38 (dp38), but not JNK, phosphorylates dATF-2 and enhances dATF-2-dependent transcription. Consistent with this, injection of dATF-2 double-stranded RNA (dsRNA) into embryos did not induce the dorsal closure defects that are commonly observed in the Drosophila JNK mutant. Furthermore, expression of the dominant-negative dp38 enhanced the aberrant wing phenotype caused by expression of a dominant-negative dATF-2. Similar genetic interactions between dATF-2 and the dMEKK1-dp38 signaling pathway also were observed in the osmotic stress-induced lethality of embryos. Loss of dATF-2 in Drosophila S2 cells by using dsRNA abrogated the induction of 40% of the osmotic stress-induced genes, including multiple immune response-related genes. This indicates that dATF-2 is a major transcriptional factor in stress-induced transcription. Thus, dATF-2 is critical for the p38-mediated stress response

CD34+ cells represent highly functional endothelial progenitor cells in murine bone marrow.

BACKGROUND: Endothelial progenitor cells (EPCs) were shown to have angiogenic potential contributing to neovascularization. However, a clear definition of mouse EPCs by cell surface markers still remains elusive. We hypothesized that CD34 could be used for identification and isolation of functional EPCs from mouse bone marrow. METHODOLOGY/PRINCIPAL FINDINGS: CD34(+) cells, c-Kit(+)/Sca-1(+)/Lin(-) (KSL) cells, c-Kit(+)/Lin(-) (KL) cells and Sca-1(+)/Lin(-) (SL) cells were isolated from mouse bone marrow mononuclear cells (BMMNCs) using fluorescent activated cell sorting. EPC colony forming capacity and differentiation capacity into endothelial lineage were examined in the cells. Although CD34(+) cells showed the lowest EPC colony forming activity, CD34(+) cells exhibited under endothelial culture conditions a more adherent phenotype compared with the others, demonstrating the highest mRNA expression levels of endothelial markers vWF, VE-cadherin, and Flk-1. Furthermore, a dramatic increase in immediate recruitment of cells to the myocardium following myocardial infarction and systemic cell injection was observed for CD34(+) cells comparing with others, which could be explained by the highest mRNA expression levels of key homing-related molecules Integrin β2 and CXCR4 in CD34(+) cells. Cell retention and incorporation into the vasculature of the ischemic myocardium was also markedly increased in the CD34(+) cell-injected group, giving a possible explanation for significant reduction in fibrosis area, significant increase in neovascularization and the best cardiac functional recovery in this group in comparison with the others. CONCLUSION: These findings suggest that mouse CD34(+) cells may represent a functional EPC population in bone marrow, which could benefit the investigation of therapeutic EPC biology

Sonic Hedgehog signaling regulates vascular differentiation and function in human CD34 positive cells

Identification of pivotal factors potentially present in the in situ environment and capable of influencing the function of CD34+ cells, which can be used for autologous cell therapy, is of paramount interest. SHh is one of the morphogens essential for embryonic vascular development as well as postnatal neovascularization, and the activation of SHh signaling with angiogenic and vascular differentiation responses in CD34+ cells by SHh treatment differed depending on the G-CSF treatment or the background disease. SHh enhanced the migration, proliferation, adhesion, and EPC colony forming capacities of G-CSF mobilized CD34+ cells, increasing the vasculogenic/angiogenic potential for neovascularization. An increase in the differentiation potential of CD34+ cells toward vascular lineages was demonstrated with SHh treatment involving TGFβ signaling pathway. The SHh-activated G-CSF mobilized CD34+ cells directly contributed to vascular regeneration while non-activated CD34+ cells showed a lower regenerative capacity in a mouse ischemic hindlimb model. SHh signaling regulates human CD34+ cell fate and function, and may potentiate the therapeutic effect of G-CSF mobilized CD34+ cells on ischemic diseases