Parathyroid hormone (PTH) and PTH related peptide (PTHrP) stimulate diverse physiological responses in a number of tissues by binding to the same receptor. We have previously cloned the gene encoding the mouse PTH/PTHrP receptor (PTHR), and have identified a promoter region. The first exon transcribed from this promoter contains untranslated sequence and is followed by an exon encoding signal sequence and the first amino acids of the mature polypeptide. We have now identified and characterized a second promoter region, located > 3 kb upstream of the original. Four partial cDNA clones, amplified from mouse kidney RNA by reverse transcription followed by the polymerase chain reaction, contain sequence corresponding to two previously unidentified exons composed of untranslated sequence. The second (3') of the two exons is spliced to the previously identified signal sequence exon. These cDNAs are highly homologous to the 5' end of a cDNA isolated from human kidney, strongly suggesting that the promoter region is conserved between mouse and humans. RNase protection and primer extension experiments have identified several transcriptional start sites extending over a region of approximately 100 bp. Unlike the previously identified promoter, this promoter is not (G+C)-rich. It lacks a consensus TATA element, but does contain a consensus CCAAT box. We have determined the expression patterns of both promoters by RNase protection with total and poly A+ RNA from several mouse tissues. The newly identified promoter is highly tissue specific, being strongly active in kidney and weakly active in liver, but not expressed in the other tissues studied. The previously identified (G+C)-rich promoter is expressed in all tissues studied. This indicates that the PTHR gene expression is controlled by regulatory signals specific to kidney and liver, as well as signals functioning in a wide variety of cell types. These results may provide insight into certain defects in PTH signalling found in humans.

The Runx2 transcription factor is critical for commitment to the osteoblast lineage. However, its role in committed osteoblasts and its functions during postnatal skeletogenesis remain unclear. We established a Runx2-floxed line with insertion of loxP sites around exon 8 of the Runx2 gene. Runx2 protein lacking the region encoded by exon 8 is imported into the nucleus and binds target DNA, but exhibits diminished transcriptional activity. We specifically deleted the Runx2 gene in committed osteoblasts using 2.3kb col1a-Cre transgenic mice. Surprisingly, the homozygous Runx2 mutant mice were born alive. The Runx2 heterozygous and homozygous null were grossly indistinguishable from wild-type littermates at birth. Runx2 deficiency did not alter proliferative capacity of osteoblasts during embryonic development (E18). Chondrocyte differentiation and cartilage growth in mutants was similar to wild-type mice from birth to 3 months of age. Analysis of the embryonic skeleton revealed poor calcification in homozygous mutants, which was more evident in bones formed by intramembranous ossification. Runx2 mutants showed progressive retardation in postnatal growth and exhibited significantly low bone mass by 1 month of age. Decreased bone formation was associated with decreased gene expression of osteoblast markers and impaired collagen assembly in the extracellular matrix. Consequently, Runx2 mutant bones exhibited decreased stiffness and structural integrity. By 3 months of age, bone acquisition in mutant mice was roughly half that of wild-type littermates. In addition to impaired osteoblast function, mutant mice showed markedly decreased osteoclast number and postnatal bone resorption. Taken together, functional deficiency of Runx2 in osteoblasts does not result in failed embryonic skeletogenesis, but disrupts postnatal bone formation.

The parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) is a G-protein-coupled receptor containin seven predicted transmembrane do . We have isolated and characterized recombinant bacteriophage AEMBL3 genomic clones containing the mouse PTHR gene, including 10 kilobases of the promoter region. The gene spans >32 kilobases and in divided into 15 exons, 8 of which contain the tansmembrne dans. The PTHR exons contining the predicted membrane-snning d are beterogeneous in length and three of the exon-ntron boundaries fall within putative transmembrane sequences, suggestin that the exons did not arise from duplication events. This arrangement is closely related to that of the growth hormone releasing factor receptor gene, particularly in the transmembrane region, providing strong evidence that the two genes evolved from a common precursor. Transcription is initiated principally at a series of sites over a 15-base-pair region. The proximal promoter region is highly (G+C)-rich and lacks an apparent TATA box or initiator element homologies but does contain CCGCCC motifs. The presumptive amino acid sequence of the encoded receptor is 99%, 91%, and 76% identical to those of the rat, human, and opossum receptors, respectively. There is no consensus polyadenylylation signal in the 3' untnted region. The poly(A) tail ofthe PTHR transcript begins 32 bases downstream ofa 35-base-long A-rich sequence, suggesting that this region directs polyadenylylation.The parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) is bound specifically by a conserved 34-amino acid region present in both parathyroid hormone (PTH) and PTH-related peptide (PTHrP). PTH regulates calcium and phosphate metabolism by binding to receptors expressed in kidney and bone (1-5). PTHrP was first identified as a major cause of malignancy-associated hypercalcemia (6, 7); however, its normal physiological role remains largely unknown. Whereas PTH expression is limited to the parathyroid, PTHrP is expressed in a wide variety of normal and malignant tissues and appears to act mainly in a para-or autocrine manner (8-11). The PTHR is a G-protein-coupled receptor containing seven predicted transmembrane domains (refs. 1-3 and references therein). Binding of ligand to the PTHR stimulates cAMP production, raises intracellular calcium, and increases levels of inositol 1,4,5-trisphosphate (2).The G-protein-coupled family of receptors is vast and includes receptors for peptide hormones, >100 odorants, neurotransmitters, and a number of other regulatory factors (12). Based on similarities between ligands and receptors (13, 14), the PTHR belongs to a subfamily that includes receptors for growth hormone releasing factor, vasoactive intestinal peptide, calcitonin, secretin, glucagon-like peptide, and glucagon. Genes for several mammalian adrenergic and serotonin receptors have been cloned and are intronless (15)(16)(17)(18)(19). Although the luteinizing hormone receptor contains 11 exons, the transmembrane and cytoplasmic regions of...

The Sp7/Osterix transcription factor is essential for bone development. Mutations of the Sp7 gene in humans are associated with craniofacial anomalies and osteogenesis imperfecta. However, the role of Sp7 in embryonic tooth development remains unknown. Here we identified the functional requirement of Sp7 for dentin synthesis and tooth development. Sp7-null mice exhibit craniofacial dysmorphogenesis and are completely void of alveolar bone. Surprisingly, initial tooth morphogenesis progressed normally in Sp7-null mice. Thus the formation of alveolar bone is not a prerequisite for tooth morphogenesis. Sp7 is required for mineralization of palatal tissue but is not essential for palatal fusion. The reduced proliferative capacity of Sp7-deficient ectomesenchyme results in small and misshapen teeth with randomly arranged cuboidal preodontoblasts and preameloblasts. Sp7 promotes functional maturation and polarization of odontoblasts. Markers of mature odontoblast (Col1a, Oc, Dspp, Dmp1) and ameloblast (Enam, Amelx, Mmp20, Amtn, Klk4) are barely expressed in incisors and molar tissues of Sp7-null mice. Consequently, dentin and enamel matrix are absent in the Sp7-null littermates. Interestingly, the Sp7 expression is restricted to cells of the dental mesenchyme indicating the effect on oral epithelium-derived ameloblasts is cell-nonautonomous. Abundant expression of Fgf3 and Fgf8 ligand was noted in the developing tooth of wild-type mice. Both ligands were remarkably absent in the Sp7-null incisor and molar, suggesting cross-signaling between mesenchyme and epithelium is disrupted. Finally, promoter-reporter assays revealed that Sp7 directly controls the expression of Fgf-ligands. Together, our data demonstrate that Sp7 is obligatory for the differentiation of both ameloblasts and odontoblasts but not for the initial tooth morphogenesis. © 2018 American Society for Bone and Mineral Research.

bStudies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis. D entinogenesis is the process by which dentin, the major mineralized tissue of teeth, is formed through progressive cytodifferentiation of progenitor cells to mature odontoblasts (1). Multiple layers of gene regulation, including those by microRNA (miRNA), orchestrate the physiologic process of dentinogenesis in a stage-specific manner (2). Progenitor cells, including dental papilla cells or dental follicle cells, derived from the ectomesenchyme of the cranial neural crest, differentiate into preodontoblasts and produce predentin. Predentin stimulates further differentiation of the cells it surrounds, giving rise to mature odontoblasts that produce dentin. Odontoblast secretion of dentin extracellular matrix proteins, including dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1), aids in the process of mineralization that forms primary dentin. However, the mechanisms of odontoblast-specific gene regulation by miRNA during dentinogenesis are not clearly understood.miRNAs are endogenous, noncoding RNAs implicated in posttranscriptional RNA silencing (3-9). The importance of miRNAs in skeletogenesis has been shown in mice by loss-offunction analysis of proteins involved in miRNA processing (Drosha and DGCR8), maturation (Dicer), and silencing (argonaute 2; AGO2), which revealed embryonic lethality and severe developmental defects upon loss of these proteins (10-15). Furthermore, cartilage-specific deletion of Dicer led to accelerated differentiation and subsequent cell death (11), whereas osteoblast-and osteoclast-specific deletion increased bone mass (13,16). Current studies on miRNA regulation of gene expression indicate a key role for this process in tooth development (17)(18)(19)(20) and in controlling cellular signaling (18,(21)(22)(23)(24)(25) and differentiation (2,26). However, ...

Tooth formation is a multifaceted process involving numerous interactions between oral epithelium and neural crest derived ecto-mesenchyme from morphogenesis to cytodifferentiation. The precise molecular regulator that drives the cyto-differentiation and dynamic cross-talk between the two cell types has yet to be fully understood. Runx2 along with its downstream target Sp7 are essential transcription factors for development of the mineralizing cell types. Global knockout of the Runx2 gene results in an arrest of tooth morphogenesis at the late bud stage. Like Runx2, Sp7-null mutants exhibit peri-natal lethality and are completely devoid of alveolar bone. However, the role of Sp7 in tooth development remains elusive. Here, we report the effects of Sp7 deletion on tooth formation. Surprisingly, tooth morphogenesis progresses normally until the mid bell stage in Sp7-homozygous mutants. Incisors and multi-cusped first and second molars were noted in both littermates. Thus, formation of alveolar bone is not a prerequisite for tooth morphogenesis. Tooth organs of Sp7-null however, were significantly smaller in size when compared to WT. Differentiation of both ameloblasts and odontoblasts was disrupted in Sp7-null mice. Only premature and disorganized ameloblasts and odontoblasts were noted in mutant mice. These data indicate that Sp7 is not required for tooth morphogenesis but is obligatory for the functional maturation of both ameloblasts and odontoblasts.

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