Functional Characterization of WNT7A Signaling in PC12 Cells

Abstract

WNT factors represent key mediators of many processes in animal development and homeostasis and act through a receptor complex comprised of members of the Frizzled and low density lipoprotein-related receptors (LRP). In mammals, 19 genes encoding Wingless and Int-related factor (WNTs), 10 encoding Frizzled, and 2 encoding LRP proteins have been identified, but little is known of the identities of individual Frizzled-LRP combinations mediating the effects of specific WNT factors. Additionally, several secreted modulators of WNT signaling have been identified, including at least three members of the Dickkopf family. WNT7A is a WNT family member expressed in the vertebrate central nervous system capable of modulating aspects of neuronal plasticity. Gene knock-out models in the mouse have revealed that WNT7A plays a role in cerebellar maturation, although its function in the development of distal limb structures and of the reproductive tract have been more intensely studied. To identify a receptor complex for this WNT family member, we have analyzed the response of the rat pheochromocytoma cell line PC12 to WNT7A. We find that PC12 cells are capable of responding to WNT7A as measured by increased ␤-catenin stability and activation of a T-cell factor-based luciferase reporter construct and that these cells express three members of the Frizzled family (Frizzled-2, -5, and -7) and LRP6. Our functional analysis indicates that WNT7A can specifically act via a Frizzled-5⅐LRP6 receptor complex in PC12 cells and that this activity can be antagonized by Dickkopf-1 and Dickkopf-3.Members of the WNT gene family encode structurally related secreted glycoprotein factors, modulating a vast array of processes during vertebrate and invertebrate embryonic development as well as several aspects of tissue homeostasis in the adult (1-4). In embryos, signaling by WNT factors controls the organization of the body plan during the early stages of development as well as organogenesis at later developmental stages. Postnatally, WNT signaling is involved in normal biological events such as tissue maturation and homeostasis and in several neoplastic pathologies (2, 5-8). For example, in the mammalian central nervous system (CNS) 1 WNT signal transduction is involved in neural induction and patterning in early embryogenesis (2, 4) as well as in organogenesis and neuronal homeostasis at later stages (9). In the adult, WNTs play a role in the control of neuronal plasticity and are implicated in CNS neoplasias such as medulloblastoma (10 -14). The analysis of the signaling events mediated by WNTs has uncovered at least three signal transduction pathways, each involved in the mediation of specific biological responses (2, 15). The most studied and best understood signaling cascade elicited by WNTs involves an interaction with a receptor complex comprising members of the Frizzled (FZD) class of 7-transmembrane receptors and a member of the low density lipoprotein receptor (LRP) family of single-pass membrane proteins (16). WNT interaction with its r...

Msx2 Exerts Bone Anabolism via Canonical Wnt Signaling

“…Moreover, as observed in other cell systems (21), transient coexpression of Wnt7a and Wnt7b eukaryotic expression plasmids significantly activate canonical TCF/LEF-dependent transcription in C3H10T1/2 cells (see supplemental material). The C3H10T1/2 multipotent mesenchymal cell line faithfully recapitulates the osteogenic versus adipogenic differentiation fate choices of bone marrow mesenchyme (23,28).…”

“…Murine models of diabetic medial calcification spatially resolved adventitial Msx2-expressing cells and the ALP-positive medial calcifying vascular cells that direct matrix mineralization; therefore, we deduced that Msx2-expressing cells elaborate paracrine signals that control osteogenic differentiation of neighboring progenitors (19). Indeed, we showed that conditioned media from Msx2-expressing C3H10T1/2 cells possessed a pro-osteogenic, adipostatic activity (19) resembling that of canonical Wnt ligands such as Wnt3a (20), Wnt7a (21), and Wnt10b (22,23). Molecular profiling by quantitative RTqPCR demonstrated up-regulation of canonical Wnt agonists Wnt3a and Wnt7a in aortic tissues (19).…”

“…mal Cells-Because Wnt7 protein levels were significantly affected by the Msx2Tg, and both Wnt7a and Wnt7b are agonists for canonical Wnt signaling (21,(57)(58)(59), Wnt7 family members are potential paracrine mediators of Msx2 action. Moreover, as observed in other cell systems (21), transient coexpression of Wnt7a and Wnt7b eukaryotic expression plasmids significantly activate canonical TCF/LEF-dependent transcription in C3H10T1/2 cells (see supplemental material).…”

Regulation of prostate cell growth and morphogenesis by Dickkopf-3

“…Thus, endogenous Dkk-3 inhibits prostate cell proliferation by negatively regulating the G1/S transition. Although Dkk family members were first identified as regulators of the Wnt signalling pathway (Glinka et al, 1998), there are conflicting reports on whether Dkk-3 affects Wnt/b-catenin signalling (Krupnik et al, 1999;Tsuji et al, 2000;Caricasole et al, 2003;Hoang et al, 2004). Therefore, we determined whether the negative effect of Dkk-3 on prostate cell proliferation correlated with any effects on Wnt/b-catenin signalling.…”

Uterine Wnt/β-catenin signaling is required for implantation

“…Genes coding for both Wnt molecules are expressed in the blastocyst at the time of implantation (13). Wnt5a is known not to signal through the canonical Wnt͞␤-catenin pathway, whereas Wnt 7a does signal through this pathway (14)(15)(16). Freshly collected conditioned medium from parental L cells or L cells expressing Wnt5a or Wnt7a was injected in the uteri of pseudopregnant transgenic females on the morning of day 4.…”