Martyn K. Robinson's research while affiliated with WWF United Kingdom and other places

Dickkopf (Dkk) family proteins are important regulators of Wnt signalling pathways, which play key roles in many essential biological processes. Here we report the first detailed structural and dynamics study of a full-length mature Dkk protein (Dkk4, residues 19-224), including determination of the first atomic resolution structure for the N-terminal cysteine-rich domain (CRD1) conserved among Dkk proteins. We discovered that CRD1 has significant structural homology to the Dkk C-terminal cysteine-rich domain (CRD2), pointing to multiple gene duplication events during Dkk family evolution. We also show that Dkk4 consists of two independent folded domains (CRD1 and CRD2) joined by a highly flexible, non-structured linker. Similarly, the N-terminal region preceding CRD1 and containing a highly conserved NXIR/K sequence motif was shown to be dynamic and highly flexible.We demonstrate that Dkk4 CRD2 mediates high-affinity binding to both the E1E2 region of LDL receptor-related protein 6 (LRP6 E1E2) and the Kremen1 (Krm1) extracellular domain. In contrast, the N-terminal region alone bound with only moderate affinity to LRP6 E1E2, consistent with binding via the conserved NXIR/K motif, but did not interact with Krm proteins. We also confirmed that Dkk and Krm family proteins function synergistically to inhibit Wnt signalling. Insights provided by our integrated structural, dynamics, interaction and functional studies have allowed us to refine the model of synergistic regulation of Wnt signalling by Dkk proteins. Our results indicate the potential for the formation of a diverse range of ternary complexes comprising Dkk, Krm and LRP5/6 proteins, allowing fine-tuning of Wnt-dependent signalling.

Administration of antibodies to sclerostin (Scl-Ab) has been shown to increase bone mass, bone mineral density (BMD) and bone strength by increasing bone formation and decreasing bone resorption in both animal studies and human clinical trials. In these studies, the magnitude and rate of increase in bone formation markers is attenuated upon repeat dosing with Scl-Ab despite a continuous and progressive increase in BMD. Here, we investigated whether the attenuation in the bone formation response following repeated administration of Scl-Ab was associated with increased expression of secreted antagonists of Wnt signalling and determined how the circulating marker of bone formation, P1NP, responded to single, or multiple doses, of Scl-Ab four days post-dosing. Female Balb/c mice were treated with Scl-Ab and we demonstrated that the large increase in serum P1NP observed following the first dose was reduced following administration of multiple doses of Scl-Ab. This dampening of the P1NP response was not due to a change in the kinetics of the bone formation marker response, or differences in exposure to the drug. The abundance of transcripts encoding several secreted Wnt antagonists was determined in femurs collected from mice following one or six doses of Scl-Ab, or vehicle treatment. Compared with vehicle controls, expression of SOST, SOST-DC1, DKK1, DKK2, SFRP1, SFRP2, FRZB, SFRP4 and WIF1 transcripts was significantly increased (approximately 1.5–4.2 fold) following a single dose of Scl-Ab. With the exception of SFRP1, these changes were maintained or further increased following six doses of Scl-Ab and the abundance of SFRP5 was also increased. Up-regulation of these Wnt antagonists may exert a negative feedback to increased Wnt signalling induced by repeated administration of Scl-Ab and could contribute to self-regulation of the bone formation response over time. After an antibody-free period of four weeks or more, the P1NP response was comparable to the naïve response, and a second phase of treatment with Scl-Ab following an antibody-free period elicited additional gains in BMD. Together, these data demonstrate that the rapid dampening of the bone formation response in the immediate post-dose period which occurs after repeat dosing of Scl-Ab is associated with increased expression of Wnt antagonists, and a treatment-free period can restore the full bone formation response to Scl-Ab.

Loss of the gene encoding the secreted Wnt antagonist sclerostin results in increased bone mass in humans and mice. Administration of antibodies to sclerostin (Scl-Ab) has been shown to increase bone mineral density (BMD) by increasing bone formation and decreasing bone resorption, in both animal studies and human clinical trials. In these studies, the magnitude and rate of increase in bone formation markers, and the rate of increase of BMD, diminishes upon repeat dosing with Scl-Ab despite a continuous and progressive increase in BMD. In this study, we investigated whether increased expression of secreted antagonists of Wnt signalling could be a contributory factor to the apparent attenuated response in markers of bone formation following repeat dosing of Scl-Ab. Treatment of female Balb/c mice (8–10 weeks old) with Scl-Ab for five weeks (10 mg/kg weekly, s.c.) increased DXA whole body BMD by 9.4%±2.0% compared with vehicle control. Serum P1NP, a bone formation marker, was measured 4 days after one, three or five doses of Scl-Ab. After the first dose of Scl-Ab, serum P1NP significantly increased versus vehicle control (165%±9%) whilst the increase was less pronounced after the third or fifth dose of Scl-Ab (56%±11% or 21%±6%, respectively). In order to investigate the mechanism of this attenuated response, mRNA expression of several secreted Wnt antagonists was determined in femurs collected from mice following five weeks of Scl-Ab or vehicle treatment. Expression of SOST, SOST-DC1, DKK1, DKK2, SFRP2, FRZB, SFRP4, SFRP5 and WIF1 transcripts was significantly increased (approximately 2–3.5 fold) upon Scl-Ab treatment compared with vehicle controls. Continuous administration of Scl-Ab is associated with up-regulation of several Wnt antagonists. This could represent a negative feedback to increased Wnt signalling induced by Scl-Ab and may help explain the attenuation in the bone formation response and in bone density increase over time.

3D reconstruction of multi-photon luminescence imaging of MWNT-Fab′-DTPA in brain slices. Mice were i.v. injected with MWNT-Fab′-DTPA (200 μg) and brains were isolated at 1 h after injection. Mice were perfused with DiI and 4% PFA at sacrifice. Brains were sectioned into 1 mm thick slices (z-step: 1 μm, number of optical sections: 150; λexcitation = 950 nm). Blood vessels appear in red (Dil stained) while f-MWNT appears in white.

Supplementary material.