Eusociality in snapping shrimps is associated with larger genomes and an accumulation of transposable elements.

Despite progress uncovering the genomic underpinnings of sociality, much less is known about how social living affects the genome. In different insect lineages, for example, eusocial species show both positive and negative associations between genome size and structure, highlighting the dynamic nature of the genome. Here, we explore the relationship between sociality and genome architecture in Synalpheus snapping shrimps that exhibit multiple origins of eusociality and extreme interspecific variation in genome size. Our goal is to determine whether eusociality leads to an accumulation of repetitive elements and an increase in genome size, presumably due to reduced effective population sizes resulting from a reproductive division of labor, or whether an initial accumulation of repetitive elements leads to larger genomes and independently promotes the evolution of eusociality through adaptive evolution. Using phylogenetically informed analyses, we find that eusocial species have larger genomes with more transposable elements (TEs) and microsatellite repeats than noneusocial species. Interestingly, different TE subclasses contribute to the accumulation in different species. Phylogenetic path analysis testing alternative causal relationships between sociality and genome architecture is most consistent with the hypothesis that TEs modulate the relationship between sociality and genome architecture. Although eusociality appears to influence TE accumulation, ancestral state reconstruction suggests moderate TE abundances in ancestral species could have fueled the initial transitions to eusociality. Ultimately, we highlight a complex and dynamic relationship between genome and social evolution, demonstrating that sociality can influence the evolution of the genome, likely through changes in demography related to patterns of reproductive skew.

Trigeminal neurons control immune-bone cell interaction and metabolism in apical periodontitis.

Apical periodontitis (AP) is an inflammatory disease occurring following tooth infection with distinct osteolytic activity. Despite increasing evidence that sensory neurons participate in regulation of non-neuronal cells, their role in the development of AP is largely unknown. We hypothesized that trigeminal ganglia (TG) Nav1.8+ nociceptors regulate bone metabolism changes in response to AP. A selective ablation of nociceptive neurons in Nav1.8Cre/Diphtheria toxin A (DTA)Lox mouse line was used to evaluate the development and progression of AP using murine model of infection-induced AP. Ablation of Nav1.8+ nociceptors had earlier progression of AP with larger osteolytic lesions. Immunohistochemical and RNAscope analyses demonstrated greater number of macrophages, T-cells, osteoclast and osteoblast precursors and an increased RANKL:OPG ratio at earlier time points among Nav1.8Cre/ DTALox mice. There was an increased expression of IL-1α and IL-6 within lesions of nociceptor-ablated mice. Further, co-culture experiments demonstrated that TG neurons promoted osteoblast mineralization and inhibited osteoclastic function. The findings suggest that TG Nav1.8+ neurons contribute to modulation of the AP development by delaying the influx of immune cells, promoting osteoblastic differentiation, and decreasing osteoclastic activities. This newly uncovered mechanism could become a therapeutic strategy for the treatment of AP and minimize the persistence of osteolytic lesions in refractory cases.

Demographic inference provides insights into the extirpation and ecological dominance of eusocial snapping shrimps.

Although eusocial animals often achieve ecological dominance in the ecosystems where they occur, many populations are unstable, resulting in local extinction. Both patterns may be linked to the characteristic demography of eusocial species-high reproductive skew and reproductive division of labor support stable effective population sizes that make eusocial groups more competitive in some species, but also lower effective population sizes that increase susceptibility to population collapse in others. Here, we examine the relationship between demography and social organization in Synalpheus snapping shrimps, a group in which eusociality has evolved recently and repeatedly. We show using coalescent demographic modeling that eusocial species have had lower but more stable effective population sizes across 100,000 generations. Our results are consistent with the idea that stable population sizes may enable competitive dominance in eusocial shrimps, but they also suggest that recent population declines are likely caused by eusocial shrimps' heightened sensitivity to environmental changes, perhaps as a result of their low effective population sizes and localized dispersal. Thus, although the unique life histories and demography of eusocial shrimps have likely contributed to their persistence and ecological dominance over evolutionary time scales, these social traits may also make them vulnerable to contemporary environmental change.

Podoplanin is dispensable for mineralized tissue formation and maintenance in the Swiss outbred mouse background.

Podoplanin, PDPN, is a mucin-type transmembrane glycoprotein widely expressed in many tissues, including lung, kidney, lymph nodes, and mineralized tissues. Its function is critical for lymphatic formation, differentiation of type I alveolar epithelial lung cells, and for bone response to biomechanical loading. It has previously been shown that Pdpn null mice die at birth due to respiratory failure emphasizing the importance of Pdpn in alveolar lung development. During the course of generation of Pdpn mutant mice, we found that most Pdpn null mice in the 129S6 and C57BL6/J mixed genetic background die at the perinatal stage, similar to previously published studies with Pdpn null mice, while all Pdpn null mice bred with Swiss outbred mice survived. Surviving mutant mice in the 129S6 and C57BL6/J mixed genetic background showed alterations in the osteocyte lacunocanalicular network, especially reduced osteocyte canaliculi in the tibial cortex with increased tibial trabecular bone. However, adult Pdpn null mice in the Swiss outbred background showed no overt differences in their osteocyte lacunocnalicular network, bone density, and no overt differences when challenged with exercise. Together, these data suggest that genetic variations present in the Swiss outbred mice compensate for the loss of function of PDPN in lung, kidney, and bone.

BMP2 expression in the endocardial lineage is required for AV endocardial cushion maturation and remodeling.

Distal outgrowth, maturation and remodeling of the endocardial cushion mesenchyme in the atrioventricular (AV) canal are the essential morphogenetic events during four-chambered heart formation. Mesenchymalized AV endocardial cushions give rise to the AV valves and the membranous ventricular septum (VS). Failure of these processes results in several human congenital heart defects. Despite this clinical relevance, the mechanisms governing how mesenchymalized AV endocardial cushions mature and remodel into the membranous VS and AV valves have only begun to be elucidated. The role of BMP signaling in the myocardial and secondary heart forming lineage has been well studied; however, little is known about the role of BMP2 expression in the endocardial lineage. To fill this knowledge gap, we generated Bmp2 endocardial lineage-specific conditional knockouts (referred to as Bmp2 cKOEndo) by crossing conditionally-targeted Bmp2flox/flox mice with a Cre-driver line, Nfatc1Cre, wherein Cre-mediated recombination was restricted to the endocardial cells and their mesenchymal progeny. Bmp2 cKOEndo mouse embryos did not exhibit failure or delay in the initial AV endocardial cushion formation at embryonic day (ED) 9.5-11.5; however, significant reductions in AV cushion size were detected in Bmp2 cKOEndo mouse embryos when compared to control embryos at ED13.5 and ED16.5. Moreover, deletion of Bmp2 from the endocardial lineage consistently resulted in membranous ventricular septal defects (VSDs), and mitral valve deficiencies, as evidenced by the absence of stratification of mitral valves at birth. Muscular VSDs were not found in Bmp2 cKOEndo mouse hearts. To understand the underlying morphogenetic mechanisms leading to a decrease in cushion size, cell proliferation and cell death were examined for AV endocardial cushions. Phospho-histone H3 analyses for cell proliferation and TUNEL assays for apoptotic cell death did not reveal significant differences between control and Bmp2 cKOEndo in AV endocardial cushions. However, mRNA expression of the extracellular matrix components, versican, Has2, collagen 9a1, and periostin was significantly reduced in Bmp2 cKOEndo AV cushions. Expression of transcription factors implicated in the cardiac valvulogenesis, Snail2, Twist1 and Sox9, was also significantly reduced in Bmp2 cKOEndo AV cushions. These data provide evidence that BMP2 expression in the endocardial lineage is essential for the distal outgrowth, maturation and remodeling of AV endocardial cushions into the normal membranous VS and the stratified AV valves.

Characterization of new bone morphogenetic protein (Bmp)-2 regulatory alleles.

Bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) is a classical morphogen; a molecule that acts at a distance and whose concentration influences cell proliferation, differentiation, and apoptosis. Key events requiring precise Bmp2 regulation include heart specification and morphogenesis and neural development. In mesenchymal cells, the concentration of BMP2 influences myogenesis, adipogenesis, chondrogenesis, and osteogenesis. Because the amount, timing, and location of BMP2 synthesis influence pattern formation and organogenesis, the mechanisms that regulate Bmp2 are crucial. A sequence within the 3'UTR of the Bmp2 mRNA termed the "ultra-conserved sequence" (UCS) has been largely unchanged since fishes and mammals diverged. Cre-lox mediated deletion of the UCS in a reporter transgene revealed that the UCS may repress Bmp2 in proepicardium, epicardium, and epicardium-derived cells (EPDC) and in tissues with known epicardial contributions (coronary vessels and valves). The UCS also repressed the transgene in the aorta, outlet septum, posterior cardiac plexus, cardiac and extra-cardiac nerves, and neural ganglia. We used homologous recombination and conditional deletion to generate three new alleles in which the Bmp2 3'UTR was altered as follows: a UCS flanked by loxP sites with or without a neomycin resistance targeting vector, or a deleted UCS. Deletion of the UCS was associated with elevated Bmp2 mRNA and BMP signaling levels, reduced fitness, and embryonic malformations.

Bone Morphogenetic Protein-2 (BMP-2) Activates NFATc1 Transcription Factor via an Autoregulatory Loop Involving Smad/Akt/Ca2+ Signaling.

Bone remodeling is controlled by dual actions of osteoclasts (OCs) and osteoblasts (OBs). The calcium-sensitive nuclear factor of activated T cells (NFAT) c1 transcription factor, as an OC signature gene, regulates differentiation of OCs downstream of bone morphogenetic protein-2 (BMP-2)-stimulated osteoblast-coded factors. To analyze a functional link between BMP-2 and NFATc1, we analyzed bones from OB-specific BMP-2 knock-out mice for NFATc1 expression by immunohistochemical staining and found significant reduction in NFATc1 expression. This indicated a requirement of BMP-2 for NFATc1 expression in OBs. We showed that BMP-2, via the receptor-specific Smad pathway, regulates expression of NFATc1 in OBs. Phosphatidylinositol 3-kinase/Akt signaling acting downstream of BMP-2 also drives NFATc1 expression and transcriptional activation. Under the basal condition, NFATc1 is phosphorylated. Activation of NFAT requires dephosphorylation by the calcium-dependent serine/threonine phosphatase calcineurin. We examined the role of calcium in BMP-2-stimulated regulation of NFATc1 in osteoblasts. 1,2Bis(2aminophenoxy)ethaneN,N,N',N'-tetraacetic acid acetoxymethyl ester, an inhibitor of intracellular calcium abundance, blocked BMP-2-induced transcription of NFATc1. Interestingly, BMP-2 induced calcium release from intracellular stores and increased calcineurin phosphatase activity, resulting in NFATc1 nuclear translocation. Cyclosporin A, which inhibits calcineurin upstream of NFATc1, blocked BMP-2-induced NFATc1 mRNA and protein expression. Expression of NFATc1 directly increased its transcription and VIVIT peptide, an inhibitor of NFATc1, suppressed BMP-2-stimulated NFATc1 transcription, confirming its autoregulation. Together, these data show a role of NFATc1 downstream of BMP-2 in mouse bone development and provide novel evidence for the presence of a cross-talk among Smad, phosphatidylinositol 3-kinase/Akt, and Ca(2+) signaling for BMP-2-induced NFATc1 expression through an autoregulatory loop.

Signatures of positive selection and local adaptation to urbanization in white-footed mice (Peromyscus leucopus).

Urbanization significantly alters natural ecosystems and has accelerated globally. Urban wildlife populations are often highly fragmented by human infrastructure, and isolated populations may adapt in response to local urban pressures. However, relatively few studies have identified genomic signatures of adaptation in urban animals. We used a landscape genomic approach to examine signatures of selection in urban populations of white-footed mice (Peromyscus leucopus) in New York City. We analysed 154,770 SNPs identified from transcriptome data from 48 P. leucopus individuals from three urban and three rural populations and used outlier tests to identify evidence of urban adaptation. We accounted for demography by simulating a neutral SNP data set under an inferred demographic history as a null model for outlier analysis. We also tested whether candidate genes were associated with environmental variables related to urbanization. In total, we detected 381 outlier loci and after stringent filtering, identified and annotated 19 candidate loci. Many of the candidate genes were involved in metabolic processes and have well-established roles in metabolizing lipids and carbohydrates. Our results indicate that white-footed mice in New York City are adapting at the biomolecular level to local selective pressures in urban habitats. Annotation of outlier loci suggests selection is acting on metabolic pathways in urban populations, likely related to novel diets in cities that differ from diets in less disturbed areas.

Removal of SOST or blocking its product sclerostin rescues defects in the periodontitis mouse model.

Understanding periodontal ligament (PDL) biology and developing an effective treatment for bone and PDL damage due to periodontitis have been long-standing aims in dental medicine. Here, we first demonstrated by cell lineage tracing and mineral double-labeling approaches that murine PDL progenitor cells display a 2- and 3-fold higher mineral deposition rate than the periosteum and endosteum at the age of 4 weeks, respectively. We next proved that the pathologic changes in osteocytes (Ocys; changes from a spindle shape to round shape with a >50% reduction in the dendrite number/length, and an increase in SOST) are the key pathologic factors responsible for bone and PDL damage in periostin-null mice (a periodontitis animal model) using a newly developed 3-dimensional FITC-Imaris technique. Importantly, we proved that deleting the Sost gene (a potent inhibitor of WNT signaling) or blocking sclerostin function by using the mAb in this periodontitis model significantly restores bone and PDL defects (n = 4-5; P < 0.05). Together, identification of the key contribution of the PDL in normal alveolar bone formation, the pathologic changes of the Ocys in periodontitis bone loss, and the novel link between sclerostin and Wnt signaling in the PDL will aid future drug development in the treatment of patients with periodontitis.

Urbanization shapes the demographic history of a native rodent (the white-footed mouse, Peromyscus leucopus) in New York City.

How urbanization shapes population genomic diversity and evolution of urban wildlife is largely unexplored. We investigated the impact of urbanization on white-footed mice,Peromyscus leucopus,in the New York City (NYC) metropolitan area using coalescent-based simulations to infer demographic history from the site-frequency spectrum. We assigned individuals to evolutionary clusters and then inferred recent divergence times, population size changes and migration using genome-wide single nucleotide polymorphisms genotyped in 23 populations sampled along an urban-to-rural gradient. Both prehistoric climatic events and recent urbanization impacted these populations. Our modelling indicates that post-glacial sea-level rise led to isolation of mainland and Long Island populations. These models also indicate that several urban parks represent recently isolated P. leucopus populations, and the estimated divergence times for these populations are consistent with the history of urbanization in NYC.

Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis.

Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages.

Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression.

Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo.

Bmp2 in osteoblasts of periosteum and trabecular bone links bone formation to vascularization and mesenchymal stem cells.

We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2-cKO(ob)) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKO(ob) mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKO(ob) osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (α-SMA, CD146 and Angiopoietin-1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in α-SMA(+) MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells.

Dkk2 has a role in terminal osteoblast differentiation and mineralized matrix formation.

Human and mouse genetic and in vitro evidence has shown that canonical Wnt signaling promotes bone formation, but we found that mice lacking the canonical Wnt antagonist Dickkopf2 (Dkk2) were osteopenic. We reaffirmed the finding that canonical Wnt signaling stimulates osteogenesis, including the differentiation from preosteoblasts to osteoblasts, in cultured osteoblast differentiation models, but we also found that canonical Wnts upregulated the expression of Dkk2 in osteoblasts. Although exogenous overexpression of Dkk before the expression of endogenous canonical Wnt (Wnt7b) suppressed osteogenesis in cultures, its expression after peak Wnt7b expression induced a phenotype resembling terminal osteoblast differentiation leading to mineralization. In addition, osteoblasts from Dkk2-null mice were poorly mineralized upon osteogenic induction in cultures, and Dkk2 deficiency led to attenuation of the expression of osteogenic markers, which could be partially reversed by exogenous expression of Dkk2. Taken together with the finding that Dkk2-null mice have increased numbers of osteoids, these data indicate that Dkk2 has a role in late stages of osteoblast differentiation into mineralized matrices. Because expression of another Wnt antagonist, FRP3, differs from Dkk2 expression in rescuing Dkk2 deficiency and regulating osteoblast differentiation, the effects of Dkk2 on terminal osteoblast differentiation may not be entirely mediated by its Wnt signaling antagonistic activity.

BMP2, but not BMP4, is crucial for chondrocyte proliferation and maturation during endochondral bone development.

The BMP signaling pathway has a crucial role in chondrocyte proliferation and maturation during endochondral bone development. To investigate the specific function of the Bmp2 and Bmp4 genes in growth plate chondrocytes during cartilage development, we generated chondrocyte-specific Bmp2 and Bmp4 conditional knockout (cKO) mice and Bmp2,Bmp4 double knockout (dKO) mice. We found that deletion of Bmp2 and Bmp4 genes or the Bmp2 gene alone results in a severe chondrodysplasia phenotype, whereas deletion of the Bmp4 gene alone produces a minor cartilage phenotype. Both dKO and Bmp2 cKO mice exhibit severe disorganization of chondrocytes within the growth plate region and display profound defects in chondrocyte proliferation, differentiation and apoptosis. To understand the mechanism by which BMP2 regulates these processes, we explored the specific relationship between BMP2 and Runx2, a key regulator of chondrocyte differentiation. We found that BMP2 induces Runx2 expression at both the transcriptional and post-transcriptional levels. BMP2 enhances Runx2 protein levels through inhibition of CDK4 and subsequent prevention of Runx2 ubiquitylation and proteasomal degradation. Our studies provide novel insights into the genetic control and molecular mechanism of BMP signaling during cartilage development.

The United States dried seahorse trade: A comparison of traditional Chinese medicine and ecommerce-curio markets using molecular identification.

Tens of millions of dried seahorses (genus Hippocampus) are traded annually, and the pressure from this trade along with their life history traits (involved parental care and small migration distances and home ranges) has led to near global population declines. This and other forms of overexploitation have led to all seahorse species being listed in Appendix II under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). The signatory nations of CITES recommended a 10-cm size limit of seahorses to ensure harvested individuals have reached reproductive maturity, and have thus had the chance to produce offspring, to maintain a more sustainable global seahorse fishery. We assessed adherence to CITES recommendations using DNA barcoding and size measurements to compare two prominent U.S. dried seahorse markets: (1) traditional Chinese medicine (TCM), and (2) non-medicinal ecommerce and coastal curio (ECC). We also estimated U.S. import abundance from CITES records. Of the nine species identified among all samples (n = 532), eight were found in the TCM trade (n = 168); composed mostly (75%) of the Indo-Pacific species Hippocampus trimaculatus, and Hippocampus spinosissimus, and the Latin American Hippocampus ingens. In contrast, ECC samples (n = 344) included 5 species, primarily juvenile Indo-Pacific Hippocampus kuda (51.5%) and the western Atlantic Hippocampus zosterae (40.7). The majority of TCM samples (85.7%) met the CITES size recommendation, in contrast to 4.8% of ECC samples. These results suggest non-size discriminatory bycatch is the most likely source of imported ECC specimens. In addition, CITES records indicate that approximately 602,275 dried specimens were imported into the U.S. from 2004-2020, but the exact species composition remains unknown as many U.S. imports records list one species or Hippocampus spp. from confiscated shipments due to difficulties in morphological identification and large numbers of individuals per shipment. Molecular identification was used to identify the species composition of confiscated shipment imports containing undesignated species, and similar to TCM, found H. trimaculatus and H. spinosissimus the most abundant. By combining DNA barcoding, size comparisons, and CITES database records, these results provide an important glimpse into the two primary dried U.S. seahorse end-markets, and may further inform the conservation status of several Hippocampus species.

Hyperglycemia and xerostomia are key determinants of tooth decay in type 1 diabetic mice.

Insulin-dependent type 1 diabetes mellitus (DM) and oral diseases are closely interrelated. Poor metabolic control in diabetics is associated with a high risk of gingivitis, periodontitis and tooth loss. Salivary flow declines in diabetics and patients suffer from xerostomia. Reduced saliva predisposes to enamel hypomineralization and caries formation; however, the mechanisms that initiate and lead to progression of tooth decay and periodontitis in type 1 DM have not been explored. To address this issue, we analyzed tooth morphology in Akita ⁻/⁻ mice that harbor a point mutation in the Ins2 insulin gene, which leads to progressive hyperglycemia. Mandibles from Akita ⁻/⁻ and wild-type littermates were analyzed by microCT, scanning EM and histology; teeth were examined for amelogenin (Amel) and ameloblastin (Ambn) expression. Mice were injected with pilocarpine to assess saliva production. As hyperglycemia may alter pulp repair, the effect of high glucose levels on the proliferation/differentiation of cultured MD10-F2 pulp cells was also analyzed. Results showed that Akita ⁻/⁻ mice at 6 weeks of age showed chalky white incisors that correlated with marked hyperglycemia and impaired saliva production. MicroCT of Akita ⁻/⁻ teeth revealed excessive enamel wearing and hypomineralization; immunostaining for Amel and Ambn was decreased. A striking feature was invasion of dentinal tubules with Streptococcus mitis and microabcesses that originated in the coronal pulp and progressed to pulp necrosis and periapical periodontitis. High levels of glucose also inhibited MD10-F2 cell proliferation and differentiation. Our findings provide the first evidence that hyperglycemia in combination with reduced saliva in a model of type1 DM leads to decreased enamel mineralization/matrix proteins and predisposes to excessive wearing and decay. Importantly, hyperglycemia adversely affects enamel matrix proteins and pulp repair. Early detection and treatment of hyperglycemia and hyposalivation may provide a useful strategy for preventing the dental complications of diabetes and promoting oral health in this population.

Reactive oxygen species derived from Nox4 mediate BMP2 gene transcription and osteoblast differentiation.

BMP-2 (bone morphogenetic protein-2) promotes differentiation of osteoblast precursor cells to mature osteoblasts that form healthy bone. In the present study, we demonstrate a novel mechanism of BMP-2-induced osteoblast differentiation. The antioxidant NAC (N-acetyl-L-cysteine) and the flavoprotein enzyme NAD(P)H oxidase inhibitor DPI (diphenyleneiodonium) prevented BMP-2-stimulated alkaline phosphatase expression and mineralized bone nodule formation in mouse 2T3 pre-osteoblasts. BMP-2 elicited a rapid generation of ROS (reactive oxygen species) concomitant with increased activation of NAD(P)H oxidase. NAC and DPI inhibited BMP-2-induced ROS production and NAD(P)H oxidase activity respectively. NAD(P)H oxidases display structurally similar catalytic subunits (Nox1-5) with differential expression in various cells. We demonstrate that 2T3 pre-osteoblasts predominantly express the Nox4 isotype of NAD(P)H oxidase. To extend this finding, we tested the functional effects of Nox4. Adenovirus-mediated expression of dominant-negative Nox4 inhibited BMP-2-induced alkaline phosphatase expression. BMP-2 promotes expression of BMP-2 for maintenance of the osteoblast phenotype. NAC and DPI significantly blocked BMP-2-stimulated expression of BMP2 mRNA and protein due to a decrease in BMP2 gene transcription. Dominant-negative Nox4 also mimicked this effect of NAC and DPI. Our results provide the first evidence for a new signalling pathway linking BMP-2-stimulated Nox4-derived physiological ROS to BMP-2 expression and osteoblast differentiation.

Matrix-bound Cyr61/CCN1 is required to retain the properties of the bone marrow mesenchymal stem cell niche but is depleted with aging.

Previously, we showed that extracellular matrices (ECMs), produced ex vivo by various types of stromal cells, direct bone marrow mesenchymal stem cells (BM-MSCs) in a tissue-specific manner and recapitulate physiologic changes characteristic of the aging microenvironment. In particular, BM-MSCs obtained from elderly donors and cultured on ECM produced by young BM stromal cells showed improved quantity, quality and osteogenic differentiation. In the present study, we searched for matrix components that are required for a functional BM-MSC niche by comparing ECMs produced by BM stromal cells from "young" (≤25 y/o) versus "elderly" (≥60 y/o) donors. With increasing donor age, ECM fibrillar organization and mechanical integrity deteriorated, along with the ability to promote BM-MSC proliferation and responsiveness to growth factors. Proteomic analyses revealed that the matricellular protein, Cyr61/CCN1, was present in young, but undetectable in elderly, BM-ECM. To assess the role of Cyr61 in the BM-MSC niche, we used genetic methods to down-regulate the incorporation of Cyr61 during production of young ECM and up-regulate its incorporation in elderly ECM. The results showed that Cyr61-depleted young ECM lost the ability to promote BM-MSC proliferation and growth factor responsiveness. However, up-regulating the incorporation of Cyr61 during synthesis of elderly ECM restored its ability to support BM-MSC responsiveness to osteogenic factors such as BMP-2 and IGF-1. We next examined aging bone and compared bone mineral density and Cyr61 content of L4-L5 vertebral bodies in "young" (9-11 m/o) and "elderly" (21-33 m/o) mice. Our analyses showed that low bone mineral density was associated with decreased amounts of Cyr61 in osseous tissue of elderly versus young mice. Our results strongly demonstrate a novel role for ECM-bound Cyr61 in the BM-MSC niche, where it is responsible for retention of BM-MSC proliferation and growth factor responsiveness, while depletion of Cyr61 from the BM niche contributes to an aging-related dysregulation of BM-MSCs. Our results also suggest new potential therapeutic targets for treating age-related bone loss by restoring specific ECM components to the stem cell niche.

Axin2 controls bone remodeling through the beta-catenin-BMP signaling pathway in adult mice.

To investigate the role of Wnt-beta-catenin signaling in bone remodeling, we analyzed the bone phenotype of female Axin2-lacZ knockout (KO) mice. We found that trabecular bone mass was significantly increased in 6- and 12-month-old Axin2 KO mice and that bone formation rates were also significantly increased in 6-month-old Axin2 KO mice compared with wild-type (WT) littermates. In vitro studies were performed using bone marrow stromal (BMS) cells isolated from 6-month-old WT and Axin2 KO mice. Osteoblast proliferation and differentiation were significantly increased and osteoclast formation was significantly reduced in Axin2 KO mice. Nuclear beta-catenin protein levels were significantly increased in BMS cells derived from Axin2 KO mice. In vitro deletion of the beta-catenin gene under Axin2 KO background significantly reversed the increased alkaline phosphatase activity and the expression of osteoblast marker genes observed in Axin2 KO BMS cells. We also found that mRNA expression of Bmp2 and Bmp4 and phosphorylated Smad1/5 protein levels were significantly increased in BMS cells derived from Axin2 KO mice. The chemical compound BIO, an inhibitor of glycogen synthase kinase 3beta, was utilized for in vitro signaling studies in which upregulated Bmp2 and Bmp4 expression was measured in primary calvarial osteoblasts. Primary calvarial osteoblasts were isolated from Bmp2(fx/fx);Bmp4(fx/fx) mice and infected with adenovirus-expressing Cre recombinase. BIO induced Osx, Col1, Alp and Oc mRNA expression in WT cells and these effects were significantly inhibited in Bmp2/4-deleted osteoblasts, suggesting that BIO-induced Osx and marker gene expression were Bmp2/4-dependent. We further demonstrated that BIO-induced osteoblast marker gene expression was significantly inhibited by Osx siRNA. Taken together, our findings demonstrate that Axin2 is a key negative regulator in bone remodeling in adult mice and regulates osteoblast differentiation through the beta-catenin-BMP2/4-Osx signaling pathway in osteoblasts.