Generation and Characterization of dickkopf3 Mutant Mice

Abstract

dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity.The Dickkopf family of secreted proteins consists of four members, which share two conserved cysteine-rich domains (12,24). The hallmark of Dkk proteins is that they function as Wnt antagonists or agonists by binding to and inhibiting or activating the Wnt coreceptor LRP6 (1, 31, 45). They show regionalized expression during vertebrate embryogenesis (5,10,13,18,20,33,46). Dkk1 is the best-characterized member of the family. It acts as an embryonic head inducer, and when overexpressed it will induce extra heads in Xenopus and zebra fish (6,12,18,22,36,46). dkk1 mutant mice are embryonic lethal, and embryos lack anterior head structure and display fused digits (36). dkk2 mouse mutants are viable but show bone defects (28). Little is known about the biological role of dkk4.By a number of criteria, dkk3 appears as a divergent member of the dkk family. (i) By DNA sequence similarity, vertebrate dkk1, -2, and -4 are more related to each other than they are to dkk3 (12). (ii) Hydra has two dkk genes, one related to vertebrate dkk1, -2, and -4 (16) and one related to vertebrate dkk3 (9). This suggests an ancient phylogenetic separation between these family members, where dkk1, -2, and -4 but not dkk3 arose by gene duplication from an ancestral dkk (16). (iii) Soggy is a protein of unknown function with sequence similarity to dkk3 but not to other dkk genes (24). The similarity is most pronounced outside the two conserved Dkk cysteine-rich domains, raising the possibility that the gene arose from an ancestral dkk3 precursor. (iv) Unlike Dkk1, -2, and -4, Dkk3 does not act as a Wnt modulator (24,29,55). While all other tested Dkk proteins bind to and modulate the Wnt receptor LRP6, as well as the Dkk coreceptor Kremen, Dkk3 has no affinity to these transmembrane proteins (7,30,32,33), and no other proteins are known to interact with it.Like other dkk members, dkk3 is expressed during vertebrate development in suggestive patterns in many organs (7,33). Prominent expression of dkk3 is observed in the brain and in fibroblasts of adult rodents (17,24,34,37,56) and in the human adrenal cortex (50).

Downregulation of Dickkopf-3 disrupts prostate acinar morphogenesis through TGF-β/Smad signaling

“…Prostate epithelial cell proliferation is increased in Dkk-3-null mice Dkk3-null mice are viable and fertile and do not show any major morphological or phenotypic alterations (del Barco Barrantes et al, 2006). To determine whether Dkk-3 affects prostate development, prostate samples were obtained from mice aged 6 and 8 weeks, when prostate development is almost complete (Sugimura et al, 1986).…”

“…The details of mice deficient for Dkk-3 were described previously (del Barco Barrantes et al, 2006). Analyses were performed using male mice aged 6 and 8 weeks.…”

“…A variety of mechanisms have been proposed to account for the inhibitory effects of Dkk-3 on tumor cell proliferation in cancer of the prostate and other organs (Abarzua et al, 2005;Kashiwakura et al, 2008;Lee et al, 2009;Lodygin et al, 2005;Yue et al, 2008). Despite the tumor suppressor activities of Dkk-3, homozygous deletion of the Dkk3 gene in mice has only minor effects on blood cell counts, lung ventilation and behaviour, with no reported increase in mortality or spontaneous tumor formation (del Barco Barrantes et al, 2006;Papatriantafyllou et al, 2012). A clue to the possible role of Dkk-3 in normal tissues comes from siRNA studies in prostate epithelial cells, where Dkk-3 expression is required for formation of acini in three-dimensional (3D) Matrigel cultures (Kawano et al, 2006).…”

Function and biological roles of the Dickkopf family of Wnt modulators

“…It was suggested that an N-terminally truncated Dkk3 encodes a substrate binding subunit, 'p29', of the type II iodothyronine 5 0 -deiodinase (D2) in rat (Leonard et al, 2000). This suggestion has not been supported (a) because of the seleno-nature of all other cloned deiodinases that act without substrate binding subunits, (b) because there is poor correlation between Dkk3/p29 and the D2 expression patterns in rat brain (MonteroPedrazuela et al, 2003) and (c) since Dkk3 knockout mice have normal deiodinase and thyroid hormone status (del Barco Barrantes et al, 2006). While Dkk3 mouse mutants are viable, they show changes in the frequency of NK cells, immunoglobulin M, hemoglobin and hematocrit levels, as well as lung ventilation.…”

Dickkopf-3 function in the prostate

“…14,26 In order to determine if loss of Dkk-3 affects prostate tissue organization in vivo, we examined Dkk3 null mice, which are viable and do not show major morphological or phenotypic alterations. 27 Dkk3 null mouse prostates at six and eight weeks were mildly disorganized in some regions, CoMMentAry CoMMentAry a NEDD4 E3 ubiquitin ligase binding protein that is highly expressed in prostate cancer, 34 and that plays a role in TGF-β stimulation of breast cancer cell invasion and proliferation. 35 We therefore hypothesize that loss of Dkk-3 leads to activation of a subset of TGF-β target genes involved in stimulating cell invasion and proliferation, such as PMEPA1, rather than those involved in growth inhibition (Fig.…”