Dkk protein family

  The structurally and functionally heterogeneous family of human Dkk-related proteins is comprised  of Dkk-1, Dkk-2, Dkk-3, and Dkk-4 and  the unique, Dkk-3 related protein termed Soggy (Sgy) (Krupnik, 1999).  hDkks 1-4 contain an N-terminal signal peptide, two highly conserved cysteine-rich domains (displaying between 80% and 90% amino acid sequence similarity) in which the positions of 10 cysteine residues are highly conserved between family members. Cys-1 and Cys-2, as these domains are referred to are separated by a linker region. The linker region between Cys-1 and Cys-2 is highly variable between hDkks and it is notably larger in hDkk-1, -2 and -4 (50-55 aa) compared with Dkk-3 (12 aa). The positions of the 10 conserved cysteines in  Cys-2 are closely similar to those in proteins of the colipase family (Aravind and Koonin, 1998). Sgy,  which possesses homology to hDkk-3 but not other Dkks lacks the cysteine-rich domains. Sgy is uniquely related to Dkk-3. Members of the Dkk-related family display unique patterns of mRNA expression in human and mouse tissues, and are secreted when expressed in 293T cells. Secreted hDkk-2 and hDkk-4 undergo proteolytic processing which results in cleavage of the second  cysteine-rich domain from the full-length protein.
 

	Current knowledge on Dkk-3 function

    hDkk-3 is the most divergent of the four human Dkks and possesses an extended N-terminal domain which precedes the Cys-1 domain and an extended C-terminal region that follows Cys-2. Current knowledge on Dickkopf proteins and their function is not contradictory to a potential function of Dkk-3 in thyroid signaling. Dickkopf  antagonizes Wnt action in Xenopus (Glinka, 1998 -- Fedi, 1999) but has not been shown to bind to Wnt directly. The mechanism by which Dickkopf (Dkk) antagonizes Wnt action remains unknown (Hlsken J, Behrens J., 2000).

    Knowledge about developmental Dickkopf function has been mostly accumulated by functional assays in Xenopus. x Dkk-1 was discovered in a screen for factors capable of inducing Xenopus head formation in the context of inhibition of
bone morphogenic protein signaling (Glinka, 1998). Dkk-1 inhibits the axis induction activity of Xwnt8, consistent with
the suggestion that simultaneous inhibition of BMP and Wnt signaling is required for head formation. Xdkk-1 does not inhibit
the secondary axis induction by Xdsh (downstream of the Wnt receptor), and is thus most likely to act as an extracellular
factor that antagonizes Wnt function directly.

    The functions of Dkks-3 remain elusive. Dkk-3 (REIC) has been found to be down regulated in immortalized cells (Tsuji, 2000)- a observation which can be considered in the light of an assumed function of REIC (Reduced Expression in Immortalized Cells) as a Wnt repressor.  Dkk-3 expression has been studied by Glinka et al 1998.
 

	Functions established for Dkk-1 (Glinka, 1998)

 Dkk-1 is the best studied family member.
ISOLATION in a functional assay:
     xDkk-1 was identified in a screen for factors capable of inducing Xenopus head formation in the context of inhibition of bone morphogenetic protein signaling. A cDNA library was screened by microinjecting pools of synthetic mRNA with or without mRNA encoding the dominant-negative Bmp-2/4 receptor (tBR)13 into two opposite blastomeres of four-cell-stage embryos. tBR injection alone induces incomplete secondary embryonic axes that lack a head. Pools of mRNA were selected that were inactive when injected alone, but induced complete secondary axes when injected with tBR. Sib selection led to an individual cDNA, dkk-1, which shows no homology to genes with known function.
    Dkk-1 is expressed in the Spemann organizer, and its function is suggested to be the one of a head mesoderm inducer, supported by the following experimental evidence: it is secreted; it anteriorizes embryos and induces complete secondary axes with heads in cooperation with BMP antagonists (BMPs are known to block  neural induction and promote the formation of ventral and lateral mesoderm in Xenopus) ; it is expressed at the  right time in the endomesoderm to function as a head inducer; and antibody injections suggest that it is required for head formation.

Dkk-1 IS SECRETED: Dkk-1 can be immunoprecipitated as a protein of relative molecular mass 40,000 (Mr 40K) from the medium of oocytes injected with FLAG-tagged dkk-1 mRNA, indicating that Dkk-1 is secreted.

Dkk-1 IS POSTTRANSLATIONALLY MODIFIED: The major band in the cell extract migrates as a protein of 35K, suggesting that Dkk-1 is post-translationally modified before secretion.

Dkk-1 IS A MONOMER: Dkk-1 appears to be a monomer, as under non-reducing conditions the mobility of the proteins is  lower than the reduced form.

Dkk-1 IS POSSIBLY C-TERMINALLY PROCESSED BEFORE SECRETION: In extracts from embryos microinjected with  dkk-1 mRNA, the polyclonal antibody anti-15 recognizes both intracellular and extracellular Dkk-1, whereas the antibody anti-14 predominantly recognizes the smaller intracellular protein. This suggests that the carboxy-terminal epitope is masked or modified upon secretion.

 Dkk-1 AS A FUNCTION IN HEAD-INDUCTION: Dkk-1 anteriorizes embryos and is able to induce head structures in synergy with inhibitors of BMP signaling. Microinjected radially into all blastomeres of four-cell Xenopus embryos led to anteriorized embryos. In ventral injections no secondary axes were observed with dkk-1. However, when dkk-1 was co-injected ventrally with tBR mRNA, embryos developed complete secondary axes with two eyes, cement glands and two beating hearts. By marker-gen analysis it was shown that dkk-1 is able to induce a 'pre-neural' fate in animal-cap ectoderm.

Dkk-1 IS REQUIRED FOR HEAD-INDUCTION: An antibody-inhibition approach using polyclonal anti-peptide antibodies raised against Dkk-1 (anti-15 antibody) led reproducibly to microcephaly in 100% (n = 47) of the embryos.  Anti-15 antibody-induced microcephaly was very efficiently rescued by injection of dkk-1 mRNA (79% normal embryos, n = 114) but not by preprolactin (97% microcephaly, n = 114), and thus is specific for dkk-1.

Dkk-1 INHIBITS WNT-SIGNALING on the level of the ligand: Axis induction by Xwnt-8 is completely inhibited by Dkk-1. We found that dkk-1 is unable to rescue secondary axis formation by any of the mRNAs in the downstream Wnt-signaling.
 

	Function and characteristics of Dkk proteins

     HINTS FOR A CYS2/LIPASE FUNCTION: Sequence conservation among the Dkks is greatest within Cys-2, suggestive of a conserved function. Colipases facilitate interactions of pancreatic lipases with lipid micelles, and for this reason it has been suggested that the Cys-2 domain of the Dkks may enable the proteins to interact with lipids in order  to regulate Wnt function. . Wnt proteins are known to remain tightly associated with the cell surface ( Smolich et al.,  1993), and a putative lipid binding function may facilitate Wnt/Dkk interactions at the plasma membrane.  Interestingly, sequence similarity between Wnt-1 and the lipid binding domain of secreted phospholipase A2  has recently been noted, further suggesting that Wnts may interact directly with lipids ( Reichsman et al., 1999).

    Dkks function EXTRACELLULARY:  The secretion of transiently overexpressed flag epitope-tagged forms of Dkks has been studied in 293T cells. hDkk-1 was secreted and migrated with a molecular mass of approx. 42-50 kDa . hDkk-3 was also secreted by 293T cells and migrated as a heterogeneous band of 45-65 kDa. Soluble hDkk-4 was detected as three major immunoreactive species of approx. 40 kDa [form (i)], 30-32 kDa [form (ii)] and 15-17 kDa [form (iii)]. The heterogeneous profiles suggestive of post-translational modification.

    GLYCOSYLATION on transiently overexpressed flag epitope-tagged forms of Dkks in 293T cells: Four potential sites of N-linked glycosylation in hDkk-3 are conserved in chick and mouse Dkk-3 . These sites are not conserved in other Dkk family members. Studied the effect of N-glycanase treatment on the mobilities of secreted hDkk-1, hDkk-3, hDkk-4 and hSgy. Soluble hDkk-3 protein displayed a substantial increase in mobility following N-glycanase treatment. The major 45-65 kDa form of soluble hDkk-3 was observed as two species of 45-55 and 40 kDa following deglycosylation. The reason for the heterogeneity of deglycosylated hDkk-3 is unclear, although it may reflect either proteolytic processing or incomplete removal of carbohydrate from one or more attachment sites. Shg displayed a  decrease in apparent molecular mass after N-Glycanase treatment, consistent with the presence of two potential sites of N-glycosylation. Taken together, these results suggest that hDkk-3 and hSgy are expressed and secreted by 293T cells as glycoproteins. hDkk-1 displays minimal, if any, attachment of N-linked carbohydrate and hDkk-4 shows no evidence of such modification.

    PROTEOLYSIS: Each hDkk possesses several potential sites of proteolytic cleavage by furin-type proteases (Nakayama, 1997), suggesting that the proteins may be subject to post-translational proteolytic processing. hDkk-4 is proteolytically processed by 293T cells, potentially by furin type proteases (Nakayama, 1997). By inference from the hDkk-4 protein sequences. The N-terminal sequence of band (i) was found to be XVLDFNNIRS, which corresponds to the sequence that follows the predicted signal peptide cleavage site (between Ala-18 and Leu-19). The band (iii) N-terminal sequence was found to be SQGRKGQEGS, which corresponds to the Cys-2 domain cleaved at the dibasic site Lys132/Lys133 (Lys 114/Lys115 of mature hDkk4). It is likely that the major 15-17 kDa form of hDkk-2 detected in these experiments also corresponds to the Cys-2 domain, although the precise site of cleavage remains to be determined.

     TISSUE-DISTRIBUTION BY NORTHERN BLOT ANALYSIS: A 1.8 kb hDkk-1 mRNA was detected in human placenta, but not in other tissues tested. hDkk-2 transcripts of approx. 4 and 4.5 kb were detected in multiple tissues,    including heart, brain, skeletal muscle and lung. A 2.5 kb hDkk-3 transcript was also widely expressed in human tissues, although levels of this mRNA were substantially higher in heart, brain and spinal cord relative to other tissues tested. hDkk-4 mRNA was undetectable in all adult and fetal human tissues examined by Northern analysis, although a survey of a human cDNA library panel by PCR with hDkk-4-specific PCR primers generated products from libraries prepared from cerebellum, activated human T-lymphocytes, lung and esophagus. A 1 kb Soggy mRNA was expressed in mouse testis, but not in other adult murine tissues tested.

     TISSUE-DISTRIBUTION BY IN SITU HYBRIDIZATION: mDkk-3 expression was seen in the adult murine brain, eye, and heart. mDkk-3 expression in the brain was found in neurons of the cortex and hippocampus. mSgy mRNA  was expressed at high levels in adult testis in the spermatogenic epithelium of the seminiferous tubules and the spermatogonia at various stages of development.
 
 

	INTRODUCTION to Thyroid hormones

     Song S, 2000:

     GENERAL: Thyroid hormones (TH) have profound effects on the growth, development and metabolism of many  tissues (Brent, 1994). In thyroid hormone metabolism, deiodination is most important because of its role in the regulation of thyroid hormone bioactivity ( Leonard and Visser). Thyroxine (T4) is converted by 5'-deiodination at the outer ring (5'D) to the bioactive hormone 3,3',5-triiodothyronine (T3) or by 5-deiodination at the inner ring (5D) to the inactive metabolite 3,3',5'-triiodothyronine (rT3). Biochemical studies have identified three types of iodothyronine deiodinases, type I (D1), type II (D2) and type III (D3) ( Kohrle; Leonard and Visser). D1, which catalyzes the 5'D and/or 5D, is highly expressed in the liver, kidney and thyroid. D1 is considered to produce the majority of circulating T3. D2, which has only 5'D activity, is expressed predominantly in the brain, anterior pituitary, brown adipose tissue and placenta. D2 plays a key role in producing T3 locally. D3, which has only 5D activity, is found predominantly in the brain, neonatal skin, placenta and fetal intestine. D3 catalyzes the inactivation of T4 and T3. Molecular cloning of D1, D2 and D3 in various species revealed that all of them are selenoproteins, which have selenocysteine(s) inserted at a specific TGA codon.

     BRENT, 2000:

     NONGENOMIC ACTIONS OF THYROID HORMONE: have long been recognized, although the specific targets and mechanisms that mediate these actions have been difficult to demonstrate (Davis, 1996). The focus of most investigations into thyroid hormone action, however, has been on the nuclear actions of thyroid hormone (Brent, G. A. The molecular basis of thyroid hormone action. N. Engl. J. Med. 331: 847-853, 1994). Nongenomic actions are characterized by onset within minutes, rather than the hours required for genomic actions. Another important and consistent difference between genomic and nongenomic actions has been the thyroid hormone metabolic product required for a response. The nuclear TR has a much higher affinity for triiodothyronine (T3) than any other analog, and thyroxine (T4) has almost no measurable action. For nongenomic effects, T4 is often more active than T3. Other thyroid hormone analogs that have been thought to be completely inactive biologically, such as reverse T3 and T2, have been shown to be active in some nongenomic effects. The biological processes that are regulated by nongenomic actions are varied and include cellular respiration, cell morphology, vascular tone, and ion homeostasis (Davis, 1996). The cellular targets include the plasma membrane, cytoskeleton, sarcoplasmic reticulum, mitochondria, and contractile elements of vascular smooth muscle. The tissues that have been the subject of most intense study include the nervous system, vascular smooth muscle, and ion transport in the red blood cell. One well described nongenomic effect of thyroid hormone is regulation of the enzyme 5'-deiodinase type II (D2), which is important for conversion of T4 to T3, especially in the brain and pituitary. Thyroid hormone inhibits D2 by a nongenomic pathway, stimulating actin-based endocytosis at the synapse and internalization of D2 from the cell surface to the perinuclear space (Leonard, J. L., and A. P. Farwell. Thyroid hormone-regulated actin polymerization in brain. Thyroid 7: 147-151, 199.)
 
 

	INTRODUCTION to Type II Iodothyronine 5'-deionidase

     GENERAL: Tissue-specific activation and inactivation of ligands of nuclear receptors which belong to the steroid retinoid-thyroid hormone superfamily (all should be considered in connection with Dkk-3) of transcription factors represents an important principle of development- and tissue-specific local modulation of hormone action. The type 2 5'-deiodinase (D2) appears to play an important role in maintaining the intracerebral T3 content relatively constant during changes in thyroidal state. Previous studies have demonstrated that the regulation of this enzyme by  thyroid hormone and its analogs occurs are posttranslational level. Three monodeiodinase isoenzymes which are involved in activation the 'prohormone' L-thyroxine (T4), the main secretory product of the thyroid gland, have been identified, characterized, and cloned. Both, type I and type II 5'-deiodinase generate the thyromimetically active hormone 3,3',5-triiodothyronine (T3) by reductive deiodination of the phenolic ring of T4. Inactivation of T4 and its product T3 occurs by deiodination of iodothyronines at the tyrosyl ring. This reaction is catalyzed both the type III 5-deiodinase and also by the type I enzyme, which has a broader substrate specificity. The three deiodinases appear to constitute a newly discovered family of selenocysteine-containing proteins and the presence of selenocysteine in the protein is critical for enzyme activity. Whereas the selenoenzyme characteristics of the type I and typeIII deiodinases are definitively established some controversy still exists for the type II 5'-deiodinase in mammals (1999). The mRNA probably encoding the type II 5'-deiodinase subunit is markedly longer than those of the two other deiodinases and its selenocysteine-insertion element is located more than 5 kB downstream of the UGA-codon in the 3'-untranslated region.

          Song S, 2000:

     mD2 mRNA TISSUE DISTRIBUTION by northern blot: Transcripts of 7.9 and 6.9 kb were detected in the brain. This pattern was similar to that of human and rat D2 (Croteau et al., 1996). Only a7.9 kb transcript was observed in the placenta and mammary gland. A fain ttranscript, 7.9 kb in length and one of 6-8 kb in length was detected in kidney and heart, respectively.

     REGULATION: cAMP analogs have been shown to induce D2 activity and/or its mRNA in mouse neuroblastoma (Gavin et al.,1990), rat pituitary ( Kim et al., 1998) and human skeletal muscle cells( Hosoi et al., 1999). Recently, a CRE was found in the 5'-upstream region of hdio2 at a similar position to that of the CRE in mdio2. The CRE accounted for basal promoter activity as well as the induction of hdio2 expression by protein kinase C (Bartha et al., 2000).

     CANCER IMPLICATION: The present results demonstrate, for the first time, that DII mRNA as well as DII activity are expressed in brain tumors, and that DII mRNA is significantly correlated with DII activity in those tissues (2000).
 
 
 

	The rDkk-3 rD2 interaction

     Leonard, 2000:

     COMPOSITION OF type II 5'-deiodinase: one p29 subunit + one 60kD subunit + a cAMP-induced activation protein + one or more unidentified catalytic subunits.

     D2: Is a membrane-bound deiodination enzyme. Generates up to 75 % of the T3 found within brain-cells (by deiodinationof T4). Brain is very short-lived in vivo. The influence of selenium on D2 catalysis is unsettled: decrease in selenium intake that nearly eliminates D1 has only marginal effects on D2 activity.

     EXPRESSION CLONING: p29 polyclonal antibodies (raised against purified p29) were used to screen a lambda zapII cDNA library. All the isolated plaques coded for the same cDNA species (!reliability). Cell-free translation was used to confirm the starting M.

     IN VIVO PROTEIN VARIANTS: By alternative ATG usage a 29-30 kD and a 27 kD protein are formed in vivo.

     TISSUE DISTRIBUTION by Northern Blotting: found in tissues expressing D2: brain and dibutyryl cAMP stimulated astrocytes.

     TISSUE DISTRIBUTION by antibody detection: Antibody was raised against the C-terminus of the p29 protein. A    29-30 kD and a 27 kD band were found in tissues expressing high levels of native D2 :hypothyroid cerebral cortex, hypothyroid BAT (dibutyryl cAMP treated astrocytes, and untreated astrocytes). P29 is primarily expressed in neurons in vivo.

     IMMUNODEPLETION endogenous: P29 is a subunit of native D2: Domain-specific anti-p29 was used to deplete p29 from detergent soluble D2 preparations. The catalytic activity of D2 was precipitated by the anti-p29 antibody (in extracts from dibutyryl cAMP stimulated astrocytes, hypothyroid cerebral cortex, hypothyroid BAT).

    P29 INFLUENCE ON D2 ACTIVITY : OVEREXPRESSION STUDIES by Adenovirus Ad5-GFP, Ad5-p29GFP: Previous studies show that generation of a catalytically active D2 in astrocytes requires p29 and one or more cAMP induced proteins. This is confirmed: p29 expression alone does not elevate D2 activity , p29 +cAMP stimulation does (stimulation is dependent on p29: immunodepletion abolishes activatory role). Quantity of functional D2 was directly correlated with quantity of the p29GFP fusion protein synthesized.

     THE CLONED P29 BINDS AN ALKYLATING ANALOG OF THE SUBSTRAT BINDING SUBUNIT OF D2: Radioactively labeled BrAcT4 was associated with p29 as shown in a pull down assay.

    P29 INFLUENCE ON D2 ACTIVITY : IN VIVO: By introduction of a the Ad5-GFP or Ad5-p29GFP in either cerebral hemisphere of neonatal rats. Ad5-p29GFP did increase native D2 activity in the treated hemisphere.

     SUBSTRATES: T4, rT3.
 
 

	p29= Dkk3 function

          General: Destination of the internalized hormone and the possibility that different modes of entry might lead to differences in targeting to intracellular locations.

          T4 binding accelerates endocytosis of p29 protein and redirects it to endosomes instead of lysosomes. Eventually p29 is recycled to the plasma membrane, presumably no longer carrying the hormone. The T4 undergoes 5' deiodination, mainly by the typeII deiodinase as in other cells of the CNS. This deiodination, which produces the more active hormone, T3, proceeds at a faster rate when T4 is diminished as a result of inactivation or degradation of the deiodinase when the cellis exposed to T4. This could be the way that cells in the nervous system regulate the intracellular production of T3 from T4, even while they accumulate T3 from plasma by another entry pathway. Whether T4 or T3 transported by any of the entry mechanisms equilibrates with the cytosolic hormone pool or is sequestered in a cytoplasmic organelle remains an open question.

          Farwell, 1989:

     D2 and its substrat-binding subunit p29 were firstly identified by affinity labeling with BrAcT4. Intact cells labeled with BrAc[125I]T4 showed three prominent radiolabeled bands of proteins of Mr 55,000, 27,000, and 18,000 (p55, p27, p18, respectively) which incorporated approximately 80% of the affinity label. All three affinity-labeled proteins were membrane associated. One protein (p27) increased 5-6-fold after treating the cells for 16 h with dibutyryl cAMP; maximal specific in corporation of affinity label into the stimulated p27 was approximately 2 pmol/mg of cell protein in intact cells. Alterations in the steady state levels of 5'D-II resulted in parallel changes in the quantity of p27. In cell sonicates, the rate of enzyme inactivation by BrAcT4 equaled the rate of affinity-label incorporation into stimulated p27, whereas p55 and p18 showed little or no specific dibutyryl cAMP-stimulated labeling. Enzyme substrates T4 and 3,3'5'-triiodothyronine (rT3) specifically blocked p27 labeling, whereas T3 and the competitive 5'D-II inhibitor EMD 21388 (a synthetic flavonoid) were much less effective. Iopanoate, an inhibitor of all deiodinase isozymes, was  ineffective in blocking p27 labeling. Inhibition kinetics revealed that iopanoate was a noncompetitive inhibitor of dibutyryl cAMP-stimulated glial cell 5'D-II, suggesting that it interacts at a site distant from the substrate-binding site. These data identify a cAMP-inducible membrane-associated protein (p27) that has many of the properties of 5'D-II.

     Safran, 1996:

     p29, the substrate-binding subunit of 5D-II, is constitutively expressed and resides, along with other 5D-II components, in membrane vesicles located in the perinuclear space of unstimulated astrocytes. Cyclic nucleotides induce the appearance of catalytically active 5D-II coincident with translocation of p29 to the plasma membrane. In addition, a cAMP-activating factor(s) is synthesized and becomes associated with the other enzyme components that are stored in vesicles located in the perinuclear space. The identification of p29 as the substrate-binding subunit of 5D-II was based  upon multiple criteria, including a direct proportionality between the quantity of BrAcT4-labeled p29 and 5D-II activity and the reciprocal relationship between inactivation and p29 labeling. In fact, the rate of enzyme inactivation is identical to the rate of affinity labeling of the p29 subunit.
 
 

	p29=Dkk3 and D2 in migration

     Leonard, 1997:

     Thyroid hormones play an important role in the growth and development of the brain. Central to the proper integration of neuronal circuitry is the ability of the growing neurite to interpret guidance cues during its migration. The action cytoskeleton is especially rich in the growth cone, and is a likely target for thyroid hormone regulation. This brief review summarizes work showing that thyroxin, but not T3, dynamically regulates the polymerization of the actin cytoskeleton in astrocytes. The ability of T4 to enhance actin polymerization, without directly affecting gene  expression, has a profound effect on the ability of the cell to interact with laminin, the major extracellular matrix protein in the developing brain. T4 also regulates the formation of key cell contacts with extracellular matrix guidance cues. These processes are likely to participate in thyroid hormone's regulation of brain development.

      Farwell AP, 1999: