The calcitriol analogue EB1089 impairs alveolarization and induces localized regions of increased fibroblast density in neonatal rat lung.

The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3, or calcitriol), is a potent mitogen for fibroblasts cultured from rat lungs at postnatal day 4 (P4), during the peak of septation (P3 to P7). In light of the key role of fibroblasts in alveolar septation, the authors conducted studies to measure the extent to which 1,25-(OH)2D3 affects lung maturation in vivo, as well as its ability to influence the stimulatory activity of all-trans retinoic acid (RA). To identify a calcitriol analogue with maximal mitogenic activity and low systemic toxicity, two compounds with reduced calcemic activity (EB1089 and CB1093) and a superagonist (MC1288) were evaluated in neonatal rat lung fibroblast cultures. All 3 analogues were more potent mitogens than 1,25-(OH)(2)D3 itself (MC1288 approximately CB1093 > EB1089 > 1,25-(OH)2D3). In addition, each was more effective than 1,25-(OH)2D3(EB1089 > CB1093 > MC1288 > 1,25-(OH)2D3) in the activation of a vitamin D response element from the platelet-derived growth factor (PDGF)-A gene, whose expression is essential for normal alveolarization. Daily administration of EB1089 to rats 4 to 12 days of age caused an increase in mean alveolar chord length (P < .0001), and also elicited prominent regions of fibroblast hypercellularity, as defined in terms of a vimentin-positive, factor VIII-negative phenotype. EB1089 and RA each induced the expression of 2 important lung structural proteins, collagen and elastin. Regions of fibroblast hypercellularity induced by EB1089 were strongly positive for expression of the alveolarization-relevant growth factors, PDGF-AA and vascular endothelial growth factor (VEGF). These studies demonstrate that 1,25-(OH)2D3 disrupts the overall alveolarization process in the neonatal lung, although it stimulates expression of some proteins associated with lung morphogenesis.

Increased hematopoietic stem cell mobilization in aged mice.

Hematopoietic stem and progenitor cells (HSPCs) are located in the bone marrow in close association with a highly organized 3-dimensional structure formed by stroma cells, referred to as the niche. Mobilization of HSPCs from bone marrow to peripheral blood in response to granulocyte colony-stimulating factor (G-CSF) requires de-adhesion of HSPCs from the niche. The influence of aging of HSPCs on cell-stroma interactions has not been determined in detail. Using a mouse model of G-CSF-induced mobilization, we demonstrated that the ability to mobilize hematopoietic stem cells is approximately 5-fold greater in aged mice. Competitive mobilization experiments confirmed that enhanced mobilization ability was intrinsic to the stem cell. Enhanced mobilization efficiency of primitive hematopoietic cells from aged mice correlated with reduced adhesion of hematopoietic progenitor cells to stroma and with elevated levels of GTP-bound Cdc42. These results might indicate that stroma-stem cell interactions are dynamic over a lifetime and result in physiologically relevant changes in the biology of primitive hematopoietic cells with age.

A 5'-distal enhanceosome in the PDGF-A gene is activated in choriocarcinoma cells via ligand-independent binding of vitamin D receptor and constitutive jun kinase signaling.

Overexpression of platelet-derived growth factor A-chain (PDGF-A) is clearly linked to autocrine and paracrine stimulation of malignant growth in many human cancers. We have shown previously that PDGF-A overexpression in choriocarcinoma, hepatoma and lung carcinoma cell lines is driven by the activity of a 66 bp enhancer element (ACE66) located approximately 7 kb upstream of the PDGF-A transcription start site. In this study, the ACE66 element is shown to be activated in JEG-3 choriocarcinoma cells through synergistic interactions between consensus DNA motifs for binding of vitamin D receptor, AP1 and ELK1. Binding of the vitamin D/retinoid-X receptor (VDR/RXRalpha) heterodimer to the ACE66 element was reconstituted in vitro with recombinant VDR/RXRalpha and with JEG-3 nuclear extract, and was verified in living JEG-3 cells by chromatin immunoprecipitation analysis. Transcriptional activity of the ACE66 element, as well as occupancy of the element by VDR/RXRalpha, was shown to be independent of stimulation with the hormonal VDR ligand, 1,25-dihydroxyvitamin D3. The jun kinase pathway of mitogen-activated protein kinase (MAPK) signaling was shown to activate the ACE66 enhancer, most likely through activation of factors binding to the AP1 element. These results identify a novel mechanism of transcriptional enhancement involving ligand-independent activity of the VDR/RXR heterodimer and MAPK signaling pathways that appears to play an important role in the overexpression of PDGF in many different settings of human malignancy.

NM23-H1 and NM23-H2 repress transcriptional activities of nuclease-hypersensitive elements in the platelet-derived growth factor-A promoter.

The platelet-derived growth factor (PDGF)-A promoter is regulated by a number of GC-rich regulatory elements that possess non-B-form DNA structures. Screening of a HeLa cDNA expression library with the C-rich strand of a PDGF-A silencer sequence (5'-S1 nuclease-hypersensitive site (SHS)) yielded three cDNA clones encoding NM23-H1, a protein implicated as a suppressor of metastasis in melanoma and breast carcinoma. Recombinant human NM23-H1 cleaved within the 3'-portions of both 5'-SHS strands in either single-stranded or duplex forms. In contrast, NM23-H2, known as a transcriptional activator with a DNA cleavage function, cleaved within the 5'-portions of both strands, revealing that NM23-H1 and NM23-H2 cleave at distinct sites of the 5'-SHS and by different mechanisms. NM23-H1 and NM23-H2 also cleaved within the PDGF-A basal promoter region, again exhibiting preferences for cleavage within the 5'- and 3'-portions of the element, respectively. Transient transfection analyses in HepG2 cells revealed that both NM23-H1 and -H2 repressed transcriptional activity driven by the PDGF-A basal promoter (-82 to +8). Activity of the negative regulatory region (-1853 to -883), which contains the 5'-SHS, was also inhibited modestly by NM23-H1 and NM23-H2. These studies demonstrate for the first time that NM23-H1 interacts both structurally and functionally with DNA. They also indicate a role for NM23 proteins in repressing transcription of a growth factor oncogene, providing a possible molecular mechanism to explain their metastasis-suppressing effects.