A recent study proposed that differentiation of dopaminergic neurons requires a conserved "dopamine motif" (DA-motif) that functions as a binding site for ETS DNA binding domain transcription factors. In the mammalian olfactory bulb (OB), the expression of a set of five genes [including tyrosine hydroxylase (Th)] that are necessary for differentiation of dopaminergic neurons was suggested to be regulated by the ETS-domain transcription factor ER81 via the DA-motif. To investigate this putative regulatory role of ER81, expression levels of these five genes were compared in both olfactory bulbs of adult wild-type mice subjected to unilateral naris closure and the olfactory bulbs of neonatal Er81 wild-type and mutant mice. These studies found that ER81 was necessary only for Th expression and not the other cassette genes. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) experiments showed that ER81 bound directly to a consensus binding site/DA-motif in the rodent Th proximal promoter. However, the ER81 binding site/DA-motif in the Th proximal promoter is poorly conserved in other mammals. Both ChIP assays with canine OB tissue and EMSA experiments with the human Th proximal promoter did not detect ER81 binding to the Th DA-motif from these species. These results suggest that regulation of Th expression by the direct binding of ER81 to the Th promoter is a species-specific mechanism. These findings indicate that ER81 is not necessary for expression of the OB dopaminergic gene cassette and that the DA-motif is not involved in differentiation of the mammalian OB dopaminergic phenotype.

Type I collagen, a major extracellular component of the periodontal ligament (PDL), is post-translationally modified by a series of specific enzymes. Among the collagen-modifying enzymes, lysyl oxidase (LOX) is essential to initiate collagen cross-linking and lysyl hydroxylases (LHs) to regulate the cross-linking pathways that are important for tissue specific mechanical properties. The purpose of this study was to investigate the effects of mechanical loading on the expression of collagen-modifying enzymes and subsequent tissue changes in PDL. Primary human PDL cells were subjected to mechanical loading in a 3D collagen gel, and gene expression and collagen component were analyzed. Wistar rats were subjected to excessive occlusal loading with or without intra-peritoneal injection of a LOX inhibitor, β-aminopropionitrile (BAPN). Upon mechanical loading, gene expression of LH2 and LOX was significantly elevated, while that of COL1A2 was not affected on hPDL-derived cells. The mechanical loading also elevated formation of collagen α-chain dimers in 3D culture. The numbers of LH2 and LOX positive cells in PDL were significantly increased in an excessive occlusal loading model. Notably, an increase of LH2-positive cells was observed only at the bone-side of PDL. Intensity of picrosirius red staining was increased by excessive occlusal loading, but significantly diminished by BAPN treatment. These results demonstrated that mechanical loading induced collagen maturation in PDL by up-regulating collagen-modifying enzymes and subsequent collagen cross-linking which are important for PDL tissue maintenance. J. Cell. Physiol. 231: 926-933, 2016. © 2015 Wiley Periodicals, Inc.

The mechanisms underlying dopamine (DA) phenotypic differentiation in the olfactory bulb (OB) have not yet been fully elucidated and are the subject of some controversy. OB DA interneurons destined for the glomerular layer were shown to originate in the subventricular zone (SVZ) and in the rostral migratory stream (RMS). The current study investigated whether calcium/calmodulin-dependent protein kinase IV (CaMKIV) either alone or together with the Ets transcription factor ER81 was necessary for phenotypic determination during migration of progenitors. In most brain areas, including the OB, CaMKIV and ER81 displayed a reciprocal distribution. In the SVZ, only ER81 could be demonstrated. In the RMS, a subpopulation of progenitors contained ER81, but few, if any, contained CaMKIV. In OB, CaMKIV expression, restricted to deep granule cells, showed limited overlap with ER81. ER81 expression was weak in deep granule cells. Strong labeling occurred in the mitral and glomerular layers, where ER81 colabeled dopaminergic periglomerular cells that expressed either tyrosine hydroxylase (TH) or green fluorescent protein, the latter reporter gene under control of 9-kb of 5' TH promoter. Odor deprivation resulted in a significant 5.2-fold decline in TH immunoreactivity, but ER81 exhibited a relatively small 1.7-fold decline in immunoreactivity. TH expression as well as brain and bulb size were unchanged in CaMKIV knockout mice. These data suggest that ER81 may be required but is not sufficient for DA neuron differentiation and that CaMKIV is not directly involved in TH gene regulation.

Defining the molecular mechanisms that underlie development and maintenance of neuronal phenotypic diversity in the CNS is a fundamental challenge in developmental neurobiology. The vast majority of olfactory bulb (OB) interneurons are GABAergic and this neurotransmitter phenotype is specified in migrating neuroblasts by transcription of either or both glutamic acid decarboxylase 1 (Gad1) and Gad2. A subset of OB interneurons also co-express dopamine, but transcriptional repression of tyrosine hydroxylase (Th) suppresses the dopaminergic phenotype until these neurons terminally differentiate. In mature OB interneurons, GABA and dopamine levels are modulated by odorant-induced synaptic activity-dependent regulation of Gad1 and Th transcription. The molecular mechanisms that specify and maintain the GABAergic and dopaminergic phenotypes in the OB are not clearly delineated. In this report, we review previous studies and present novel findings that provide insight into the contribution of epigenetic regulatory mechanisms for controlling expression of these neurotransmitter phenotypes in the OB. We show that HDAC enzymes suppress the dopaminergic phenotype in migrating neuroblasts by repressing Th transcription. In the mature interneurons, both Th and Gad1 transcription levels are modulated by synaptic activity-dependent recruitment of acetylated histone H3 on both the Th and Gad1 proximal promoters. We also show that HDAC2 has the opposite transcriptional response to odorant-induced synaptic activity when compared to Th and Gad1. These findings suggest that HDAC2 mediates, in part, the activity-dependent chromatin remodeling of the Th and Gad1 proximal promoters in mature OB interneurons.

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Fibrillar type I collagen, the predominant organic component in bone, is stabilized by lysyl oxidase (LOX)-initiated covalent intermolecular cross-linking, an important determinant of bone quality. However, the impact of collagen cross-linking on the activity of bone cells and subsequent tissue remodeling is not well understood. In this study, we investigated the effect of collagen cross-linking on bone cellular activities employing a loss-of-function approach, using a potent LOX inhibitor, β-aminopropionitrile (BAPN). Osteoblastic cells (MC3T3-E1) were cultured for 2 weeks in the presence of 0–2 mM BAPN to obtain low cross-linked collagen matrices. The addition of BAPN to the cultures diminished collagen cross-links in a dose-dependent manner and, at 1 mM level, none of the major cross-links were detected without affecting collagen production. After the removal of cellular components from these cultures, MC3T3-E1, osteoclasts (RAW264.7), or mouse primary bone marrow-derived stromal cells (BMSCs) were seeded. MC3T3-E1 cells grown on low cross-link matrices showed increased alkaline phosphatase (ALP) activity. The number of multinucleate tartrate-resistant acid phosphatase (TRAP)-positive cells increased in RAW264.7 cells. Initial adhesion, proliferation, and ALP activity of BMSCs also increased. In the animal experiments, 4-week-old C57BL/6 mice were fed with BAPN-containing diet for 8 weeks. At this point, biochemical analysis of bone demonstrated that collagen cross-links decreased without affecting collagen content. Then, the diet was changed to a control diet to minimize the direct effect of BAPN. At 2 and 4 weeks after the change, histological samples were prepared. Histological examination of femur samples at 4 weeks showed a significant increase in the number of bone surface osteoblasts, while the bone volume and surface osteoclast numbers were not significantly affected. These results clearly demonstrated that the extent of collagen cross-linking of bone matrix affected the differentiation of bone cells, underscoring the importance of collagen cross-linking in the regulation of cell behaviors and tissue remodeling in bone. Characterization of collagen cross-linking in bone may be beneficial to obtain insight into not only bone mechanical property, but also bone cellular activities.

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Gamma-amino-butyric acid (GABA) regulates the proliferation and migration of olfactory bulb (OB) interneuron progenitors derived from the subventricular zone (SVZ), but the role of GABA in the differentiation of these progenitors has been largely unexplored. This study examined the role of GABA in the differentiation of OB dopaminergic interneurons using neonatal forebrain organotypic slice cultures prepared from transgenic mice expressing GFP under the control of the tyrosine hydroxylase (Th) gene promoter (ThGFP). KCl-mediated depolarization of the slices induced ThGFP expression. The addition of GABA to the depolarized slices further increased GFP fluorescence by inducing ThGFP expression in an additional set of periglomerular cells. These findings showed that GABA promoted differentiation of SVZ-derived OB dopaminergic interneurons and suggested that GABA indirectly regulated Th expression and OB dopaminergic neuron differentiation through an acceleration of the maturation rate for the dopaminergic progenitors. Additional studies revealed that the effect of GABA on ThGFP expression required activation of L- and P/Q-type Ca+2 channels as well as GABAA and GABAB receptors. These voltage-gated Ca+2 channels and GABA receptors have previously been shown to be required for the co-expressed GABAergic phenotype in the OB interneurons. Together, these findings suggest that Th expression and the differentiation of OB dopaminergic interneurons are coupled to the co-expressed GABAergic phenotype, and demonstrate a novel role for GABA in neurogenesis.