Functional Characterization of WNT7A Signaling in PC12 Cells

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 β-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-5·LRP6 receptor complex in PC12 cells and that this activity can be antagonized by Dickkopf-1 and Dickkopf-3.


SUMMARY
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 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 signalling have been identified including at least three members of the Dickkopf family. WNT7A is a WNT family member expressed in the vertebrate CNS 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. In order to identify a receptor complex for this WNT family member, we have analysed 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 TCF-based luciferase reporter construct, and that these cells express three members of the Frizzled family (Frizzled-2, Frizzled-5 and Frizzled-7) and LRP6. Our functional analysis indicates that WNT7A can specifically act via a Frizzled-5/LRP6 receptor complex in PC12 cells, and that this activity can be antagonized by Dickkopf-1 and Dickkopf-3.

INTRODUCTION
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)(2)(3)(4). In embryos, signalling by WNT factors controls the organization of the body plan during the early stages of development as well as organogenesis at later developmental stages. Post-natally, WNT signalling 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) 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)(11)(12)(13)(14). The analysis of the signalling 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 signalling 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 receptor results in an increase in the stability of catenin, whose accumulation results in translocation to the nucleus where it can interact with members of the TCF class of transcription factors and therefore modulate gene expression. The stability of catenin is controlled by WNT through the modulation of a large cytoplasmic protein complex comprised of the proteins AXIN, APC, GBP/FRAT and GSK3 , this latter controlling directly the level of catenin phosphorylation and its consequent degradation by the proteasome pathway these genes are expressed in a temporally and spatially regulated fashion, often in overlapping domains, while others are more widely expressed (19)(20)(21). These observations suggest a degree of specificity of action of the individual ligands and of relative affinities in the ligand-receptor interactions. Given the wider expression patterns of LRP genes and the tissue-specificity of expression displayed by many of the more numerous FZD genes, the specificity of the cellular response to individual WNT ligands is likely to depend largely on the individual FZD proteins expressed on the cell membrane.   2B). This latter mutation represents a human allele serendipitously isolated during the amplification of full length WNT7A cDNA (35) analogous to that associated with the postaxial hemimelia mutation in the mouse, which was predicted to encode a non-functional protein (46). The data presented here demonstrate that the px WNT7A allele does indeed encode a non-functional product.

WNT7A signals via the canonical pathway in PC12 cells
Interestingly, while WNT7A can activate the canonical pathway in PC12 cells, it is a rather inefficient inducer of a Ca ++ -dependent NFAT-reporter construct (data not shown), suggesting that it cannot stimulate the Ca ++ -dependent WNT pathway in this system. Two LRPs can function as co-receptors for FZD proteins, namely LRP5 and LRP6 (16). An RT-PCR analysis revealed that only LRP6 is expressed in PC12 cells (Fig. 5A)  These results suggest that DKK3 can also act as a negative modulator of WNT signalling.
Moreover, the data indicate that DKK2 can act as a positive modulator of WNT signalling (as previously reported), and that this activity may be mediated by LRP5. As for the other expression plasmids, the amount of protein produced by the three DKK expression constructs in PC12 cells was assayed by Western blotting using commercial antibodies specific for each DKK protein.
Unfortunately, we were unable to detect any signal. As a functional response to DKK expression was observed, we believe the inability to detect DKK protein expression from the three plasmids was not due to lack of protein expression but rather to a technical problem associated with the use of the anti-DKK antibodies.

U n t r a n s f e c t e d + W n t 7 A -F Z D 2 U n t r a n s f e c t e d + W n t 7 A -F Z D 5 U n t r a n s f e c t e d + W n t 7 A -F Z D 7
A C B Fig  Fig. 3 . 3

U n t r a n s f e c t e d + W n t 7 A -F Z D 2 U n t r a n s f e c t e d + W n t 7 A -F Z D 5 U n t r a n s f e c t e d + W n t 7 A -F Z D 7
A C B Fig Fig. 4        Samples RLU B L R P 5 L R P 6 -a c t i n L R P 5 L R P 6 -a c t i n