Species-specific sensitivity to TGFß signaling and changes to the Mmp13 promoter underlie avian jaw development and evolution.

Precise developmental control of jaw length is critical for survival, but underlying molecular mechanisms remain poorly understood. The jaw skeleton arises from neural crest mesenchyme (NCM), and we previously demonstrated that these progenitor cells express more bone-resorbing enzymes including Matrix metalloproteinase 13 (Mmp13) when they generate shorter jaws in quail embryos versus longer jaws in duck. Moreover, if we inhibit bone resorption or Mmp13, we can increase jaw length. In the current study, we uncover mechanisms establishing species-specific levels of Mmp13 and bone resorption. Quail show greater activation of and sensitivity to transforming growth factor beta (TGFß) signaling than duck; where intracellular mediators like SMADs and targets like Runt-related transcription factor 2 (Runx2), which bind Mmp13, become elevated. Inhibiting TGFß signaling decreases bone resorption, and overexpressing Mmp13 in NCM shortens the duck lower jaw. To elucidate the basis for this differential regulation, we examine the Mmp13 promoter. We discover a SMAD-binding element and single nucleotide polymorphisms (SNPs) near a RUNX2-binding element that distinguish quail from duck. Altering the SMAD site and switching the SNPs abolish TGFß sensitivity in the quail Mmp13 promoter but make the duck promoter responsive. Thus, differential regulation of TGFß signaling and Mmp13 promoter structure underlie avian jaw development and evolution.

miR-433-3p suppresses bone formation and mRNAs critical for osteoblast function in mice.

MicroRNAs (miRNAs) are key posttranscriptional regulators of osteoblastic commitment and differentiation. miR-433-3p was previously shown to target Runt-related transcription factor 2 (Runx2) and to be repressed by bone morphogenetic protein (BMP) signaling. Here, we show that miR-433-3p is progressively decreased during osteoblastic differentiation of primary mouse bone marrow stromal cells in vitro, and we confirm its negative regulation of this process. Although repressors of osteoblastic differentiation often promote adipogenesis, inhibition of miR-433-3p did not affect adipocyte differentiation in vitro. Multiple pathways regulate osteogenesis. Using luciferase-3' untranslated region (UTR) reporter assays, five novel miR-433-3p targets involved in parathyroid hormone (PTH), mitogen-activated protein kinase (MAPK), Wnt, and glucocorticoid signaling pathways were validated. We show that Creb1 is a miR-433-3p target, and this transcription factor mediates key signaling downstream of PTH receptor activation. We also show that miR-433-3p targets hydroxysteroid 11-ß dehydrogenase 1 (Hsd11b1), the enzyme that locally converts inactive glucocorticoids to their active form. miR-433-3p dampens glucocorticoid signaling, and targeting of Hsd11b1 could contribute to this phenomenon. Moreover, miR-433-3p targets R-spondin 3 (Rspo3), a leucine-rich repeat-containing G-protein coupled receptor (LGR) ligand that enhances Wnt signaling. Notably, Wnt canonical signaling is also blunted by miR-433-3p activity. In vivo, expression of a miR-433-3p inhibitor or tough decoy in the osteoblastic lineage increased trabecular bone volume. Mice expressing the miR-433-3p tough decoy displayed increased bone formation without alterations in osteoblast or osteoclast numbers or surface, indicating that miR-433-3p decreases osteoblast activity. Overall, we showed that miR-433-3p is a negative regulator of bone formation in vivo, targeting key bone-anabolic pathways including those involved in PTH signaling, Wnt, and endogenous glucocorticoids. Local delivery of miR-433-3p inhibitor could present a strategy for the management of bone loss disorders and bone defect repair. © 2021 American Society for Bone and Mineral Research (ASBMR).

Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development.

Precisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

MicroRNA-433 Dampens Glucocorticoid Receptor Signaling, Impacting Circadian Rhythm and Osteoblastic Gene Expression.

Serum glucocorticoids play a critical role in synchronizing circadian rhythm in peripheral tissues, and multiple mechanisms regulate tissue sensitivity to glucocorticoids. In the skeleton, circadian rhythm helps coordinate bone formation and resorption. Circadian rhythm is regulated through transcriptional and post-transcriptional feedback loops that include microRNAs. How microRNAs regulate circadian rhythm in bone is unexplored. We show that in mouse calvaria, miR-433 displays robust circadian rhythm, peaking just after dark. In C3H/10T1/2 cells synchronized with a pulse of dexamethasone, inhibition of miR-433 using a tough decoy altered the period and amplitude of Per2 gene expression, suggesting that miR-433 regulates rhythm. Although miR-433 does not directly target the Per2 3'-UTR, it does target two rhythmically expressed genes in calvaria, Igf1 and Hif1α. miR-433 can target the glucocorticoid receptor; however, glucocorticoid receptor protein abundance was unaffected in miR-433 decoy cells. Rather, miR-433 inhibition dramatically enhanced glucocorticoid signaling due to increased nuclear receptor translocation, activating glucocorticoid receptor transcriptional targets. Last, in calvaria of transgenic mice expressing a miR-433 decoy in osteoblastic cells (Col3.6 promoter), the amplitude of Per2 and Bmal1 mRNA rhythm was increased, confirming that miR-433 regulates circadian rhythm. miR-433 was previously shown to target Runx2, and mRNA for Runx2 and its downstream target, osteocalcin, were also increased in miR-433 decoy mouse calvaria. We hypothesize that miR-433 helps maintain circadian rhythm in osteoblasts by regulating sensitivity to glucocorticoid receptor signaling.

Variations in hepatic biomarkers in American alligators (Alligator mississippiensis) from three sites in Florida, USA.

Sub-individual biomarkers are sub-lethal biological responses commonly used in the assessment of wildlife exposure to environmental contaminants. In this study, we examined the activity of glutathione-s-transferase (GST) and lactate dehydrogenase (LDH), and metallothionein (MT) concentrations among captive-raised alligator hatchlings, wild-caught juveniles, and wild-caught adults. Juveniles and adults were collected from three locations in Florida (USA) with varying degrees of contamination (i.e. Lake Apopka (organochlorine polluted site), Merritt Island National Wildlife Refuge (NWR) (metal polluted site), and Lake Woodruff NWR (reference site)). We examined whether changes in the response of these three biomarkers were age and sex dependent or reflected site-specific variations of environmental contaminants. Juvenile alligators from Merritt Island NWR had higher MT concentrations and lower GST activity compared to those from the other two sites. This outcome was consistent with higher metal pollution at this location. Sexually dimorphic patterns of MT and GST (F > M) were observed in juvenile alligators from all sites, although this pattern was not observed in adults. GST activity was lower in captive-raised alligators from Lake Apopka and Merritt Island NWR as compared to animals from Lake Woodruff NWR, suggesting a possible developmental modulator at these sites. No clear patterns were observed in LDH activity. We concluded that GST and MT demonstrate age and sex specific patterns in the alligators inhabiting these study sites and that the observed variation among sites could be due to differences in contaminant exposure.

IGF-I 3' untranslated region: strain-specific polymorphisms and motifs regulating IGF-I in osteoblasts.

Reduced IGF-I is associated with low bone mass in humans and mice. C3H/He/J (C3H) mice have higher skeletal IGF-I and greater bone mass than C57BL/6J (B6). We hypothesized that strain-related genotypic differences in Igf1 affected skeletal function. The Igf1 coding region is nonpolymorphic, but its 3' untranslated region (UTR) is polymorphic between C3H and B6. Luciferase-Igf1 3' UTR reporter constructs showed that these polymorphic regions did not affect UTR function. IGF-I splice variants give rise to a common mature IGF-I peptide, but different E peptides. We identified two splice products, exon 4+6 (Ea) and exon 4+5+6 (Eb, mechano-growth factor) and found that their abundance was unchanged during osteoblastic differentiation. The Igf1 3' UTR encoded by exon 6 contains alternative polyadenylation sites. Proximal site use produces a short 3' UTR of approximately 195 bases, whereas distal site usage results in an approximately 6300-base UTR. Although Igf1 mRNA levels did not change during osteoblastic differentiation, distal polyadenylation site usage was increased in B6 cells but not in C3H. The resulting long Igf1 RNA isoform is less stable and has decreased translation efficiency, which may be one mechanism contributing to decreased IGF-I in B6 vs. C3H mice. Although the long UTR contains a conserved [GU](18) repeat, which is a positive regulator of UTR activity, it is also targeted by negative regulators, miR-29 and miR-365. These microRNAs are increased in B6 and C3H cells during osteoblastic differentiation. Differential expression of the long Igf1 3' UTR isoform may be a possible mechanism for enhanced IGF-I regulation in B6 vs. C3H mice.

Cadmium-induced decrease in RUNX2 mRNA expression and recovery by the antioxidant N-acetylcysteine (NAC) in the human osteoblast-like cell line, Saos-2.

Exposure to cadmium poses a threat to human health, including increased susceptibility to developing the bone disease osteoporosis. Despite its recognized importance as an environmental toxin, little is known about how cadmium directly impacts bone-forming osteoblasts. We previously reported that cadmium induces apoptosis in human osteoblast-like Saos-2 cells. In this work, we hypothesize that cadmium exposure induces oxidative stress which leads to decreased RUNX2 mRNA expression and increased apoptotic death, and predict that the antioxidant NAC mitigates the damaging effects of cadmium. Oxidative stress is implicated in osteoporosis; furthermore the osteoblast transcriptional factor RUNX2 is reported to play a protective role against osteoporosis in postmenopausal women. Cells treated with 10 microM CdCl2 exhibited signs of oxidative damage including depletion in glutathione, increased reactive oxygen species formation, and enhanced lipid peroxidation. RUNX2 mRNA expression, by RT-PCR, was significantly reduced after exposure to 10 microM CdCl2. Pretreatment with the antioxidant NAC (1mM) prevented cadmium-induced decrease in RUNX2 mRNA and protected cells from apoptotic death. This study provides insight into the mechanisms underlying cadmium-induced osteotoxicity. In addition, this study distinguishes itself by identifying RUNX2 as a target for heavy metal-induced osteotoxicity.