Structures of HCMV Trimer reveal the basis for receptor recognition and cell entry.

Human cytomegalovirus (HCMV) infects the majority of the human population and represents the leading viral cause of congenital birth defects. HCMV utilizes the glycoproteins gHgLgO (Trimer) to bind to platelet-derived growth factor receptor alpha (PDGFRα) and transforming growth factor beta receptor 3 (TGFßR3) to gain entry into multiple cell types. This complex is targeted by potent neutralizing antibodies and represents an important candidate for therapeutics against HCMV. Here, we determine three cryogenic electron microscopy (cryo-EM) structures of the trimer and the details of its interactions with four binding partners: the receptor proteins PDGFRα and TGFßR3 as well as two broadly neutralizing antibodies. Trimer binding to PDGFRα and TGFßR3 is mutually exclusive, suggesting that they function as independent entry receptors. In addition, Trimer-PDGFRα interaction has an inhibitory effect on PDGFRα signaling. Our results provide a framework for understanding HCMV receptor engagement, neutralization, and the development of anti-viral strategies against HCMV.

Osteoinductive effect of soluble transforming growth factor beta receptor 3 on human osteoblast lineage.

The development of bone requires carefully choregraphed signaling to bone progenitors to form bone. Our group recently described the requirement of transforming growth factor beta receptor 3 (TGFßR3), a receptor involved in TGFß pathway signaling, during osteoblast lineage commitment in mice. The TGFß pathway is known to play multiple osteo-inductive and osteo-inhibitory roles during osteoblast development and TGFßR3 human mutations are associated with reduced bone mineral density, making TGFßR3 a unique target for bone inductive therapy. In this article, we demonstrated increased mineralization of human pediatric bone-derived osteoblast-like cells (HBO) when treated with soluble TGFßR3 (sR3) using Alizarin Red staining. Osteogenic commitment of HBO cells was demonstrated by induction of osteogenic genes RUNX2, osteocalcin, osteopontin, and osterix. Evaluation of the canonical TGFß pathway signaling demonstrated that sR3 was able to induce bone formation in HBO cells, mainly through activation of noncanonical targets of TGFß pathway signaling including AKT, ERK, and p38 MAP kinases. Inhibition of these osteogenic noncanonical pathways in the HBO cells also inhibited mineralization, suggesting they are each required. Although no induction of SMAD1, 5, and 9 was observed, there was the activation of SMAD2 and 3 suggesting that sR3 is primarily signaling via the noncanonical pathways during osteogenic induction of the HBO. Our results highlight the important role of TGFßR3 in osteoblast induction of mineralization in human bone cells through noncanonical targets of TGFß signaling. Future studies will focus on the ability of sR3 to induce bone regeneration in vivo using animal models.

Mapping mRNA Expression of Glaucoma Genes in the Healthy Mouse Eye.

Purpose/Aim: Many genes have been associated with primary open-angle glaucoma (POAG). Knowing exactly where they are expressed in the eye helps to unravel POAG pathology and to select optimal targets for intervention. We investigated whether RNA in situ hybridization (RNA-ISH) is a convenient technique to obtain detailed pan-ocular expression data of these genes. We tested this for four diverse candidate POAG genes, selected because of unclear ocular distribution (F5 and Dusp1) and relevance for potential new therapies (Tnf, Tgfßr3). Optn, a POAG gene with well-known ocular expression pattern served as control. Methods: We made a list of candidate glaucoma genes reported in genetic studies. A table of their ocular expression at the tissue level was compiled using publicly available microarray data (the ocular tissue database). To add cellular detail we performed RNA-ISH for Optn, Tnf, Tgfßr3, F5, and Dusp1 on eyes of healthy, 2-month-old, pigmented, and albino mice. Results: Expression of the Optn control matched with published immunohistochemistry data. Ocular expression of Tnf was generally low, with patches of higher Tnf expression, superficially in the corneal epithelium. F5 had a restricted expression pattern with high expression in the nonpigmented ciliary body epithelium and moderate expression in the peripapillary region. Tgfßr3 and Dusp1 showed ubiquitous expression. Conclusions: RNA-ISH is a suitable technique to determine the ocular expression pattern of POAG genes, adding meaningful cellular detail to existing microarray expression data. For instance, the high expression of F5 in the nonpigmented ciliary body epithelium suggests a role of this gene in aqueous humor dynamics and intraocular pressure. In addition, the ubiquitous expression of Tgfßr3 has implications for designing TGF-ß-related glaucoma therapies, with respect to side effects. Creating pan-ocular expression maps of POAG genes with RNA-ISH will help to identify POAG pathways in specific cell types and to select targets for drug development.

BMP2 rescues deficient cell migration in Tgfbr3(-/-) epicardial cells and requires Src kinase.

During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types which contribute to the coronary vessels. The type III transforming growth factor-ß receptor (TGFßR3) is required for epicardial cell invasion and development of coronary vasculature in vivo. Bone Morphogenic Protein-2 (BMP2) is a driver of epicardial cell migration. Utilizing a primary epicardial cell line derived from Tgfbr3(+/+) and Tgfbr3(-/-) mouse embryos, we show that Tgfbr3(-/-) epicardial cells are deficient in BMP2 mRNA expression. Tgfbr3(-/-) epicardial cells are deficient in 2-dimensional migration relative to Tgfbr3(+/+) cells; BMP2 induces cellular migration to Tgfbr3(+/+) levels without affecting proliferation. We further demonstrate that Src kinase activity is required for BMP2 driven Tgfbr3(-/-) migration. BMP2 also requires Src for filamentous actin polymerization in Tgfbr3(-/-) epicardial cells. Taken together, our data identifies a novel pathway in epicardial cell migration required for development of the coronary vessels.