Paul J. Yaworsky

Wnt/β-Catenin Signaling Is a Normal Physiological Response to Mechanical Loading in Bone

A preliminary expression profiling analysis of osteoblasts derived from tibia explants of the high bone mass LRP5 G171V transgenic mice demonstrated increased expression of canonical Wnt pathway and Wnt/β-catenin target genes compared with non-transgenic explant derived osteoblasts. Therefore, expression of Wnt/β-catenin target genes were monitored after in vivo loading of the tibia of LRP5 G171V transgenic mice compared with non-transgenic mice. Loading resulted in the increased expression of Wnt pathway and Wnt/β-catenin target genes including Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43 in both genotypes; however, there was a further increased in transcriptional response with the LRP5 G171V transgenic mice. Similar increases in the expression of these genes (except cyclin D1) were observed when non-transgenic mice were pharmacologically treated with a canonical Wnt pathway activator, glycogen synthase kinase 3β inhibitor and then subjected to load. These in vivo results were further corroborated by in vitro mechanical loading experiments in which MC3T3-E1 osteoblastic cells were subjected to 3400 microstrain alone for 5 h, which increased the expression of Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43. Furthermore, when MC3T3-E1 cells were treated with either glycogen synthase kinase 3β inhibitor or Wnt3A to activate Wnt signaling and then subjected to load, a synergistic up-regulation of these genes was observed compared with vehicle-treated cells. Collectively, the in vivo and in vitro mechanical loading results support that Wnt/β-catenin signaling is a normal physiological response to load and that activation of the Wnt/β-catenin pathway enhances the sensitivity of osteoblasts/osteocytes to mechanical loading.

Transgenic analyses reveal developmentally regulated neuron- and muscle-specific elements in the murine neurofilament light chain gene promoter.

We report here the developmental activity of regulatory elements that reside within 1.7 kilobases of the murine neurofilament light chain (NF-L) gene promoter. NF-L promoter activity is first detected at embryonic day 8.5 in neuroepithelial cells. Neuron-specific gene expression is maintained in the spinal cord until embryonic day 12.5 and at later developmental stages in the brain and sensory neuroepithelia. After day 14.5, the promoter becomes active in myogenic cells. Transgene expression in both neurons and muscle is consistent with the detection of endogenous NF-L transcript in both neuronal and myogenic tissues of neonates by reverse transcriptase-polymerase chain reaction. Neuron- and muscle-specific activities of the NF-L promoter decrease and are nearly undetectable after birth. Thus, the 1.7-kilobase NF-L promoter contains regulatory elements for initiation but not maintenance of transcription from the NF-L locus. Deletion analyses reveal that independent regulatory elements control the observed tissue-specific activities and implicate a potential MyoD binding site as the muscle-specific enhancer. Our results demonstrate that the NF-L promoter contains distinct regulatory elements for both neuron- and muscle-specific gene expression and that these activities are temporally separated during embryogenesis.

(1-(4-(Naphthalen-2-yl)pyrimidin-2-yl)piperidin-4-yl)methanamine: A Wingless β-Catenin Agonist That Increases Bone Formation Rate

A high-throughput screening campaign to discover small molecule leads for the treatment of bone disorders concluded with the discovery of a compound with a 2-aminopyrimidine template that targeted the Wnt beta-catenin cellular messaging system. Hit-to-lead in vitro optimization for target activity and molecular properties led to the discovery of (1-(4-(naphthalen-2-yl)pyrimidin-2-yl)piperidin-4-yl)methanamine (5, WAY-262611). Compound 5 has excellent pharmacokinetic properties and showed a dose dependent increase in the trabecular bone formation rate in ovariectomized rats following oral administration.

Herpes simplex virus transcriptional activator VP16 is detrimental to preimplantation development in mice

The herpes simplex virus transactivator protein VP16 is frequently used to regulate gene expression in several experimental systems, including transgenic mice. It has been suggested that high levels of VP16 expression in mice may be lethal. In order to systematically address this issue, we linked the VP16 gene to promoters that are active early and in a variety of tissues throughout development, such as the human β‐actin promoter or the rat nestin gene enhancer. VP16 expression was assayed using a LacZ reporter gene linked to a VP16‐responsive immediate early gene promoter. We show here that expression of VP16 at high levels is detrimental to pre‐implantation development. By culturing embryos in vitro, we demonstrate that this effect is exerted at the transition from the 2‐cell to the 4‐cell stage, reducing survival to the blastocyst stage dramatically. On the other hand, transgenic mice expressing VP16 transgenes at postimplantation stages are viable. These results suggest a differential sensitivity to VP16 expression in different cell types and stages of development. The reduction of embryo survival by VP16 implicates herpes virus infection as a potential cause of infertility. Mol. Reprod. Dev. 55:37–46, 2000.

Caudal dysgenesis in islet-1 transgenic mice

Maternal diabetes during pregnancy is responsible for the occurrence of diabetic embryopathy, a spectrum of birth defects that includes heart abnormalities, neural tube defects, and caudal dysgenesis syndromes. Here, we report that mice transgenic for the homeodomain transcription factor Isl‐1 develop profound caudal growth defects that resemble human sacral/caudal agenesis. Isl‐1 is normally expressed in the pancreas and is required for pancreas development and endocrine cell differentiation. Aberrant regulation of this pancreatic transcription factor causes increased mesodermal cell death, and the severity of defects is dependent on transgene dosage. Together with the finding that mutation of the pancreatic transcription factor HLXB9 causes sacral agenesis, our results implicate pancreatic transcription factors in the pathogenesis of birth defects associated with diabetes.

Mutation of a putative nuclear receptor binding site abolishes activity of the nestin midbrain enhancer

Regional differences in gene expression are critical to the proper development of specialized cell types in the nervous system. The ventral midbrain is the prominent source of dopaminergic neurons, which are affected in Parkinsons disease. We have recently identified a gene regulatory element that is specifically active in ventral midbrain neuroepithelium of developing embryos. This 204-bp transcriptional enhancer is conserved within the second intron of mammalian nestin genes and contains a putative binding site for a protein of the nuclear receptor family. Our present study shows, by mutagenesis and reporter gene assay in transgenic mice, that this site is essential for enhancer function in the developing midbrain. The characterization of regulatory sites and transcription factors with specific activity in the ventral midbrain provides insight into the molecular mechanisms by which neural progenitor cells become specified towards particular neuronal differentiation pathways.

Hit to lead studies on (hetero)arylpyrimidines—Agonists of the canonical Wnt-β-catenin cellular messaging system

A series of (hetero)arylpyrimidines agonists of the Wnt-beta-catenin cellular messaging system have been prepared. These compounds show activity in U2OS cells transfected with Wnt-3a, TCF-luciferase, Dkk-1 and tk-Renilla. Selected compounds show minimal GSK-3beta inhibition indicating that the Wnt-beta-catenin agonism activity most likely comes from interaction at Wnt-3a/Dkk-1. Two examples 1 and 25 show in vivo osteogenic activity in a mouse calvaria model. One example 1 is shown to activate non-phosphorylated beta-catenin formation in bone.