Osteoblast-specific expression ofthe bone protein osteocalcin is controlled at the transcriptional level by the steroid hormone la,25-dihydroxyvitamin D3 Osteocalcin is an abundant osteoblast-specific noncollagenous bone protein that functions in mineralization under direct control of the vitamin D hormone la,25-dihydroxyvitamin D3[1,25(OH)2D3] (1-3). Despite the controversy surrounding its function in bone, levels of this gene product in the circulation have been correlated with increased states of bone turnover, and thus osteocalcin may represent a marker for metabolic bone disease (4-6). In view of the importance of this protein as a potential marker for bone pathophysiology, knowledge of its transcriptional regulation in osteoblasts is essential.The modulation of osteocalcin levels by hormones such as 1,25(OH)2D3 is widely believed to occur at the level of transcription (7,8). The mechanism of vitamin D3 induction likely involves the 1,25(OH)2D3 receptor (VDR), a 48-kDa protein whose recent cloning and structural characterization (9, 10) suggest a DNA-binding protein belonging to the steroid receptor gene superfamily (11). The molecular nature of this interaction, as well as that with other vitamin D3-inducible genes, however, has not been clarified. In this report, we describe regions within the osteocalcin gene promoter that contribute to transcriptional activation and then map a region that confers vitamin D response. The steroid response element-related nature of this sequence provides evidence that 1,25(OH)2D3 operates through its intracellular receptor to activate gene transcription analogously to that of other small nonpeptide hormones (12-14).

BackgroundGlaucoma is characterized by the progressive dysfunction and loss of retinal ganglion cells. Recent work in animal models suggests that a critical neuroinflammatory event damages retinal ganglion cell axons in the optic nerve head during ocular hypertensive injury. We previously demonstrated that monocyte-like cells enter the optic nerve head in an ocular hypertensive mouse model of glaucoma (DBA/2 J), but their roles, if any, in mediating axon damage remain unclear.MethodsTo understand the function of these infiltrating monocyte-like cells, we used RNA-sequencing to profile their transcriptomes. Based on their pro-inflammatory molecular signatures, we hypothesized and confirmed that monocyte-platelet interactions occur in glaucomatous tissue. Furthermore, to test monocyte function we used two approaches to inhibit their entry into the optic nerve head: (1) treatment with DS-SILY, a peptidoglycan that acts as a barrier to platelet adhesion to the vessel wall and to monocytes, and (2) genetic targeting of Itgam (CD11b, an immune cell receptor that enables immune cell extravasation).ResultsMonocyte specific RNA-sequencing identified novel neuroinflammatory pathways early in glaucoma pathogenesis. Targeting these processes pharmacologically (DS-SILY) or genetically (Itgam / CD11b knockout) reduced monocyte entry and provided neuroprotection in DBA/2 J eyes.ConclusionsThese data demonstrate a key role of monocyte-like cell extravasation in glaucoma and demonstrate that modulating neuroinflammatory processes can significantly lessen optic nerve injury.Electronic supplementary materialThe online version of this article (10.1186/s13024-018-0303-3) contains supplementary material, which is available to authorized users.

The human vitamin D receptor (VDR) has been cloned recently. Two cDNAs comprising the full-length VDR were spliced, cloned into a mammalian expression vector, and transiently expressed in COS-1 cells. The protein product exhibited properties consistent with that observed for receptor in human cells. A series of 5'- and 3'-deletions of the full-length VDR cDNA was prepared and evaluated. Native DNA binding was localized to a peptide fragment (residues 1-114) whose most prominent feature is the cysteine rich region proven to represent the DNA binding domain in other steroid receptors. Steroid binding-competence required synthesis of a peptide that initiated C-terminal to the DNA-binding domain at residue 114 and which contained the remaining 313 residues. To determine the location of elements within the receptor necessary for transcription, an osteocalcin gene promoter-chloramphenicol acetyltransferase reporter gene was cotransfected together with wild type or mutant VDR cDNAs and the latter's effect on chloramphenicol acetyltransferase activity was assessed. Cotransfection of wild type receptor alone resulted in efficient transcription of the reporter plasmid. However, synthesis of a peptide containing the DNA binding domain as well as 76 residues carboxy terminal to this region exhibited some degree of activity, albeit constitutive. These results suggest that the functional domains of the VDR are similar to that of other steroid receptors and that these domains participate in the transcriptional regulation of the human osteocalcin gene.

Over the past few decades, (poly)peptide block copolymers have been widely employed in generating well-defined nanostructures as vehicles for targeted drug delivery applications. We previously reported the assembly of thermoresponsive nanoscale vesicles from an elastin-b-collagen like peptide (ELP-CLP). The vesicles were observed to dissociate at elevated temperatures, despite the LCST-like behavior of the tethered ELP domain, which is suggested to be triggered by the unfolding of the CLP domain. Here, the potential of using the vesicles as drug delivery vehicles for targeting collagen-containing matrices is evaluated. The sustained release of an encapsulated model drug was achieved over a period of three weeks, following which complete release could be triggered via heating. The ELP-CLP vesicles show strong retention on a collagen substrate, presumably through collagen triple helix interactions. Cell viability and proliferation studies using fibroblasts and chondrocytes suggest that the vesicles are highly cytocompatible. Additionally, essentially no activation of a macrophage-like cell line is observed, suggesting that the vesicles do not initiate an inflammatory response. Endowed with thermally controlled delivery, the ability to bind collagen, and excellent cytocompatibility, these ELP-CLP nanovesicles are suggested to have significant potential in the controlled delivery of drugs to collagen-containing matrices and tissues.

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