A role for the CXCR4-CXCL12 axis in the little skate, Leucoraja erinacea

Abstract

The interaction between C-X-C chemokine receptor type 4 (CXCR4) and its cognate ligand C-X-C motif chemokine ligand 12 (CXCL12) plays a critical role in regulating hematopoietic stem cell activation and subsequent cellular mobilization. Extensive studies of these genes have been conducted in mammals, but much less is known about the expression and function of CXCR4 and CXCL12 in non-mammalian vertebrates. In the present study, we identify simultaneous expression of CXCR4 and CXCL12 orthologs in the epigonal organ (the primary hematopoietic tissue) of the little skate, Leucoraja erinacea. Genetic and phylogenetic analyses were functionally supported by significant mobilization of leukocytes following administration of Plerixafor, a CXCR4 antagonist and clinically important drug. Our results provide evidence that, as in humans, Plerixafor disrupts CXCR4/CXCL12 binding in the little skate, facilitating release of leukocytes into the bloodstream. Our study illustrates the value of the little skate as a model organism, particularly in studies of hematopoiesis and potentially for preclinical research on hematological and vascular disorders.

Keywords: C-X-C chemokine ligand 12; C-X-C chemokine receptor type 4; Plerixafor/AMD3100; elasmobranch; mobilization.

Fig. 1.

Annotated multiple alignment of vertebrate ligand C-X-C motif chemokine ligand 12 (CXCL12; subset of the vertebrate chemokine ligand multiple alignment; see Supplemental Fig. S3). The little skate sequence (CXCL12_Le) is in boldface. The portion of the little skate sequence retrieved from sequencing is written in capital letters, whereas the inferred portion is written in lowercase letters. The characteristic C-X-C motif is denoted by a boldfaced underline. Sequences involved in receptor binding are highlighted in dark gray, whereas an amino acid involved in dimerization is boxed. Sites responsible for heparin binding are italicized. The receptor activation motif (KP) is bolded and italicized. The receptor and heparin binding site (RCXCR) is highlighted in light gray and italicized. Uniformly conserved cysteines are in boldface. Individual β-strands (β1–6) and α-helices (α1–3), inferred from the human CXCL12 sequence, are denoted by arrows above the alignment. Sequences responsible for turns in the secondary protein structure in human CXCL12 are underlined. For the consensus sequence, identical amino acids are denoted by an asterisk (*), whereas those with low similarity (i.e., 50%) are denoted by a period (.) and those with high similarity (i.e., 70%) are denoted by colon (:). Under the consensus sequence, the NH₂ terminus and COOH terminus are shown as black and gray bars, respectively. The sequence accession numbers are described in Supplemental Table S1, and species abbreviations are as follows: Cm, elephant shark (Callorhincus milii); Dr, zebrafish (Danio rerio); Gg, chicken (Gallus gallus); Hs, human (Homo sapiens); Mm, mouse (Mus musculus); Lc, coelacanth (Latimeria chalumnae); Le, little skate (Leucoraja erinacea); Sc, catshark (Scyliorhinus canicula); Tr, pufferfish (Takifugu rubripes).

Fig. 2.

Annotated multiple alignment of vertebrate chemokine receptor type 4 (CXCR4; subset of the vertebrate chemokine ligand multiple alignment; see Supplemental Fig. S4). The little skate sequence (CXCR4_Le) is in boldface. The portion of the little skate sequence retrieved from sequencing is written in capital letters, whereas the inferred portion is written in lowercase letters. The characteristic C-X-C motif is denoted by a boldfaced underline. Sequences involved in chemokine binding are highlighted in black with white letters, whereas sequences involved in dimerization are highlighted in light gray. The boxed sequence (DRY for all species except lamprey) denotes an area that is critical for signaling. Uniformly conserved cysteines are in boldface. Sequences responsible for turns in the secondary protein structure in human CXCR4 are underlined. Individual β-strands (β3–6) and α-helices (α1–16) inferred from the human CXCR4 sequence are denoted by arrows above the alignment. For the consensus sequence, identical amino acids are denoted by an asterisk (*), whereas those with low similarity (i.e., 50%) are denoted by a period (.), and those with high similarity (i.e., 70%) are denoted by a colon (:). Under the consensus sequence, the NH₂ terminus and COOH terminus are shown as black bars, transmembrane domains (TM1–7) as white bars, and extracellular loops (ECL1–3) and intracellular loops (ICL1–3) as gray bars. The sequence accession numbers are described in Supplemental Table S1, and species abbreviations are as follows: Cm, elephant shark (Callorhincus milii); Dr, zebrafish (Danio rerio); Gg, chicken (Gallus gallus); Hs, human (Homo sapiens); Mm, mouse (Mus musculus); Lc, coelacanth (Latimeria chalumnae); Le, little skate (Leucoraja erinacea); Pm, lamprey (Petromyzon marinus); Sc, catshark (Scyliorhinus canicula); Tr, pufferfish (Takifugu rubripes).

Fig. 3.

Phylogenetic tree of vertebrate chemokine ligands from the C-X-C motif chemokine ligand (CXCL) 8 (pink), CXCL11 (blue), CXCL12 (green), CXCL13 (yellow), and CXCL14 (purple) gene families. The tree was constructed in the MEGA6 program (54) using a MuscleWS multiple alignment. The evolutionary history was inferred by using the maximum likelihood method based on the LG model (30) with invariable sites (+I; 6.1275% sites). A discrete γ-distribution was used to model evolutionary rate differences among sites (+G; five categories, parameter = 3.6495). Node values represent %bootstrap confidence derived from 10,000 replicates. The little skate CXCL12 sequence (CXCL12_Le) is in boldface. The analysis included 38 amino acid sequences. All positions containing gaps or missing data were eliminated, resulting in a total of 65 positions in the final data set. The sequence accession numbers are described in Supplemental Table S1, and species abbreviations are as follows: Cm, elephant shark (Callorhincus milii); Dr, zebrafish (Danio rerio); Gg, chicken (Gallus gallus); Hs, human (Homo sapiens); Mm, mouse (Mus musculus); Lc, coelacanth (Latimeria chalumnae); Le, little skate (Leucoraja erinacea); Sc, catshark (Scyliorhinus canicula); Tr, pufferfish (Takifugu rubripes).

Fig. 4.

Phylogenetic tree of vertebrate chemokine receptors from the chemokine receptor type (CXCR) 1/2 (blue), CXCR3 (pink), CXCR4 (green), CXCR5 (yellow), and CXCR6 (purple) gene families. The tree was constructed in the MEGA6 program (54) using a MuscleWS multiple alignment. The evolutionary history was inferred by using the maximum likelihood method based on the JTT mixture-based model (23) with frequencies (+F). A discrete γ-distribution was used to model evolutionary rate differences among sites (+G; 5 categories, parameter = 2.3322). Node values represent %bootstrap confidence derived from 10,000 replicates. The little skate CXCR4 sequence (CXCR4_Le) is in boldface. The analysis included 50 amino acid sequences. All positions containing gaps and missing data were eliminated, resulting in a total of 166 positions in the final data set. The sequence accession numbers are described in Supplementary Table S1, and species abbreviations are as follows: Cm, elephant shark (Callorhincus milii); Dr, zebrafish (Danio rerio); Gg, chicken (Gallus gallus); Hs, human (Homo sapiens); Mm, mouse (Mus musculus); Lc, coelacanth (Latimeria chalumnae); Le, little skate (Leucoraja erinacea); Pm, lamprey (Petromyzon marinus); Sc, catshark (Scyliorhinus canicula); Tr, pufferfish (Takifugu rubripes).

Fig. 5.

%Leukocytes pre- and post-Plerixafor injection. For all animals, the preinjection percentage was calculated by averaging the leukocyte percentages from the 3 blood draws before injection. The postinjection percentage was calculated by taking the leukocyte percentage from a single blood draw at 3 different end points: 4 h postinjection for the control group (n = 3), 2 h postinjection for 1 experimental group (n = 3), and 6 h postinjection for the other experimental group (n = 3). An ANCOVA comparing the 3 groups on postinjection leukocyte percentages after controlling for baseline (preinjection) leukocyte percentages was statistically significant [F(2,5) = 6.33, P = 0.04], with a large effect size (partial η² = 0.72). Planned orthogonal comparisons found no significant difference in the no. of mobilized leukocytes between the 2 experimental groups postinjection (P = 0.93), but the experimental animals had significantly higher leukocyte percentages than control animals postinjection (P = 0.04).