Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells

Several biological events are controlled by Hedgehog (Hh) signaling, including osteoblast phenotype development. This study aimed at evaluating the gene expression profile of human mesenchymal stem cells (hMSCs) treated with the Hh agonist, purmorphamine, focusing on Hh signaling and osteoblast differentiation. hMSCs from bone marrow were cultured in non‐osteogenic medium with or without purmorphamine (2 µM) for periods of up to 14 days. Purmorphamine up‐regulated gene expression of the mediators of Hh pathway, SMO, PTCH1, GLI1, and GLI2. The activation of Hh pathway by purmorphamine increased the expression of several genes (e.g., RUNX2 and BMPs) related to osteogenesis. Our results indicated that purmorphamine triggers Hh signaling pathway in hMSCs, inducing an increase in the expression of a set of genes involved in the osteoblast differentiation program. Thus, we conclude that Hh is a crucial pathway in the commitment of undifferentiated cells to the osteoblast lineage. J. Cell. Biochem. 113: 204–208, 2012. © 2011 Wiley Periodicals, Inc.     

A Group of ent-Kaurane Diterpenoids Inhibit Hedgehog Signaling and Induce Cilia Elongation

The Hedgehog (Hh) signaling pathway plays important roles in the tumorigenesis of multiple cancers and is a key target for drug discovery. In a screen of natural products extracted from Chinese herbs, we identified eight ent-Kaurane diterpenoids and two triterpene dilactones as novel Hh pathway antagonists. Epistatic analyses suggest that these compounds likely act at the level or downstream of Smoothened (Smo) and upstream of Suppressor of Fused (Sufu). The ent-Kauranoid-treated cells showed elongated cilia, suppressed Smo trafficking to cilia, and mitotic defects, while the triterpene dilactones had no effect on the cilia and ciliary Smo. These ent-Kaurane diterpenoids provide new prototypes of Hh inhibitors, and are valuable probes for deciphering the mechanisms of Smo ciliary transport and ciliogenesis.     

Developmental signaling: Does it bridge the gap between cilia dysfunction and renal cystogenesis?

For more than a decade, evidence has accumulated linking dysfunction of primary cilia to renal cystogenesis, yet molecular mechanisms remain undefined. The pathogenesis of renal cysts is complex, involving multiple cellular aberrations and signaling pathways. Adding to this complexity, primary cilia exhibit multiple roles in a context‐dependent manner. On renal epithelial cells, primary cilia act as mechanosensors and trigger extracellular Ca²⁺ influx in response to laminar fluid flow. During mammalian development, primary cilia mediate the Hedgehog (Hh), Wnt, and Notch pathways, which control cell proliferation and differentiation, and tissue morphogenesis. Further, experimental evidence suggests the developmental state of the kidney strongly influences renal cystic disease. Thus, we review evidence for regulation of Ca²⁺ and cAMP, key molecules in renal cystogenesis, at the primary cilium, the role of Hh, Wnt, and Notch signaling in renal cystic disease, and the interplay between these developmental pathways and Ca²⁺ signaling. Indeed if these developmental pathways influence renal cystogenesis, these may represent novel therapeutic targets that can be integrated into a combination therapy for renal cystic disease. Birth Defects Research (Part C) 102:159–173, 2014. © 2014 Wiley Periodicals, Inc.     

Endothelial primary cilia inhibit atherosclerosis

Primary cilia are microtubule‐based structures present on most mammalian cells that are important for intercellular signaling. Cilia are present on a subset of endothelial cells where they project into the vessel lumen and are implicated as mechanical sensors of blood flow. To test the in vivo role of endothelial cilia, we conditionally deleted Ift88, a gene required for ciliogenesis, in endothelial cells of mice. We found that endothelial primary cilia were dispensable for mammalian vascular development. Cilia were not uniformly distributed in the mouse aorta, but were enriched at vascular branch points and sites of high curvature. These same sites are predisposed to the development of atherosclerotic plaques, prompting us to investigate whether cilia participate in atherosclerosis. Removing endothelial cilia increased atherosclerosis in Apoe^?/? mice fed a high‐fat, high‐cholesterol diet, indicating that cilia protect against atherosclerosis. Removing endothelial cilia increased inflammatory gene expression and decreased eNOS activity, indicating that endothelial cilia inhibit pro‐atherosclerotic signaling in the aorta.     

The mir‐7 and bag of marbles genes regulate Hedgehog pathway signaling in blood cell progenitors in Drosophila larval lymph glands

Hedgehog (Hh) pathway signaling is crucial for the maintenance of blood cell progenitors in the lymph gland hematopoietic organ present in Drosophila third instar larvae. Previous studies from our lab have likewise shown the importance of the mir‐7 and bag of marbles (bam) genes in maintaining the progenitor state. Thus, we sought to investigate a possible interaction between the Hh pathway and mir‐7/bam in the prohemocyte population within this hematopoietic tissue. Gain of function mir‐7 was able to rescue a blood cell progenitor depletion phenotype caused by Patched (Ptc) inhibition of Hh pathway signaling in these cells. Similarly, expression of a dominant/negative version of Ptc was able to rescue the severe reduction of prohemocytes due to bam loss of function. Furthermore, we demonstrated that Suppressor of fused [Su(fu)], another known inhibitor of Hh signaling, likely serves as a translational repression target of the mir‐7 miRNA. Our results suggest the mir‐7/bam combination regulates the Hh signaling network through repression of Su(fu) to maintain hemocyte progenitors in the larval lymph gland.     

Let-7d modulates the proliferation,migration, tubulogenesis of endothelial cells

Calcitriol, vitamin D3 (VD3), and structurally related VD3 analogues are inhibitors of Hh signaling in multiple contexts and are promising anti-cancer agents in Hh-dependent forms of cancer; however, the cellular mechanisms through which these compounds regulate Hh signal transmission are not clearly defined. Previous studies in this area have implicated both Smoothened, a key mediator of Hh signaling, and the vitamin D receptor (VDR) as potential mediators of Hh inhibition for this class of seco-steroids. We have performed a series of in vitro studies to more fully probe the cellular mechanisms that govern seco-steroid-mediated inhibition of Hh signaling. Our results support a role for both the Hh and VDR pathways in this process, as well as the possibility that other, as yet unidentified proteins, are also central to seco-steroid-mediated inhibition of Hh signaling.

     

Competition between two phosphatases fine-tunes Hedgehog signaling

Hedgehog (Hh) signaling is essential for embryonic development and adult homeostasis. How its signaling activity is fine-tuned in response to fluctuated Hh gradient is less known. Here, we identify protein phosphatase V (PpV), the catalytic subunit of protein phosphatase 6, as a homeostatic regulator of Hh signaling. PpV is genetically upstream of widerborst (wdb), which encodes a regulatory subunit of PP2A that modulates high-level Hh signaling. We show that PpV negatively regulates Wdb stability independent of phosphatase activity of PpV, by competing with the catalytic subunit of PP2A for Wdb association, leading to Wdb ubiquitination and subsequent proteasomal degradation. Thus, regulated Wdb stability, maintained through competition between two closely related phosphatases, ensures graded Hh signaling. Interestingly, PpV expression is regulated by Hh signaling. Therefore, PpV functions as a Hh activity sensor that regulates Wdb-mediated PP2A activity through feedback mechanisms to maintain Hh signaling homeostasis.     

The contrasting roles of primary cilia and cytonemes in Hh signaling

Hedgehog (Hh) is a paracrine signaling protein with major roles in development and disease. In vertebrates and invertebrates, Hh signal transduction is carried out almost entirely by evolutionarily conserved components, and in both, intercellular movement of Hh is mediated by cytonemes – specialized filopodia that serve as bridges that bring distant cells into contact. A significant difference is the role of the primary cilium, a slender, tubulin-based protuberance of many vertebrate cells. Although the primary cilium is essential for Hh signaling in cells that have one, most Drosophila cells lack a primary cilium. This perspective addresses the roles of primary cilia and cytonemes, and proposes that for Hh signaling, the role of primary cilia is to provide a specialized hydrophobic environment that hosts lipid-modified Hh and other components of Hh signal transduction after Hh has traveled from elsewhere in the cell. Implicit in this model is the idea that initial binding and uptake of Hh is independent of and segregated from the processes of signal transduction and activation.