Promotion of Bone Morphogenetic Protein Signaling by Tetraspanins and Glycosphingolipids

<div><p>Bone morphogenetic proteins (BMPs) belong to the transforming growth factor β (TGFβ) superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the <i>C</i>. <i>elegans</i> BMP-like “Sma/Mab” signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development.</p></div

Interactions among TSP-12, TSP-14, TSP-21 and the receptors SMA-6 and DAF-4.

<p>Diploid yeast cells expressing specific Cub- and Nub- fusion proteins were grown in SC-Ade,-His, Trp,-Leu,-Ura (SC-AHTLU) plates supplemented with 0.3mM of methionine. Cub and NubG were each used as negative controls. NubWT was used as a positive control to indicate expression of each Cub-PLV fusion protein. The potassium channel KAT1 was used as a control for specificity. KAT1 can interact with KAT1, but not with any of the proteins tested here.</p

The <i>sma-9(0)</i> suppressor mutations revert the M lineage dorsal-to-ventral fate transformation defect in <i>sma-9(0)</i> mutants to the wild-type pattern.

<p>(A, B) Schematic representation of the M lineage in wild-type or <i>sma-9(0);susm</i> (A), and <i>sma-9(0)</i> (B) animals. (C-D) Diagrams of an adult wild-type or <i>sma-9(0);susm</i> worm (C) and an adult <i>sma-9(0)</i> animal (D), showing the CC phenotype. <i>sma-9(0)</i> mutants lack the two M-derived CC that are present in wild-type or <i>sma-9(0);susm</i> animals (in blue arrowheads). (E and F) Merged GFP and DIC images of <i>tsp-21(jj77) sma-9(cc604)</i> (E) and <i>sma-9(cc604)</i> (F) worms carrying the <i>CC</i>::<i>gfp</i> marker at the late L4 stage. BWM: body-wall muscle, CC: coelomocyte, SM: sex myoblast. d: dorsal, v: ventral, l: left, r: right, a: anterior, p: posterior.</p

TSP-21 positively modulates LIN-12/Notch signaling in the M lineage.

<p>***<i>p</i><0.001, <i>lin-12(n676n930ts); tsp-21(jj77)</i> versus <i>lin-12(n676n930ts)</i> worms (unpaired two-tailed Student’s <i>t</i>-test).</p><p>TSP-21 positively modulates LIN-12/Notch signaling in the M lineage.</p

Rescue of the small body size of <i>tsp-21(jj77)</i> worms by tissue-specific expression of <i>tsp-21</i> cDNA.

<p>Body length was normalized to that of <i>tsp-21(jj77)</i> mutants. For each genotype, data from two transgenic lines were pooled and averaged. Data shown are mean ± s.e.m.</p><p>***<i>p</i><0.0001, different transgenic worms versus <i>tsp-21(jj77)</i> (unpaired two-tailed Student’s <i>t</i>-test).</p><p>Rescue of the small body size of <i>tsp-21(jj77)</i> worms by tissue-specific expression of <i>tsp-21</i> cDNA.</p

<i>tsp-21</i> encodes a conserved tetraspanin protein of the C6a group.

<p>(A) A schematic of the TSP-21 protein, showing the four transmembrane (TM) domains and the two extracellular loops (EC1 and EC2). The locations of the <i>jj60</i> and <i>jj77</i> molecular lesions are shown. (B) Diagrams of the <i>tsp-21</i> genomic and GFP tagged constructs. The location of the <i>tm6269</i> deletion as well as the <i>jj60</i> and <i>jj77</i> molecular lesions are shown. (C) A ClustalX sequence alignment [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005221#pgen.1005221.ref104" target="_blank">104</a>] of TSP-21 and other C6a tetraspanins from <i>Drosophila</i>, human and mouse, highlighting the four transmembrane domains (shaded light green), the conserved cysteine residues (red letters) and the amino acids mutated or deleted in <i>jj60</i> or <i>jj77</i> (red boxes). Identical residues are marked with asterisks (*) and conserved residues are marked with either colons (:) or periods (.) above the alignment. The Genbank accession numbers for the proteins shown in panel C are: <i>Drosophila</i> Tsp5D (isoform C, NP_001259266.1), human TSPAN4 (NP_001020406.1), mouse Tspan-4 (NP_001239517.1), human TSPAN9 (NP_001161792.1), mouse Tspan-9 (NP_780623.1).</p

<i>tsp-21(jj77)</i> mutants exhibit reduced RAD-SMAD reporter expression.

<p>(A-H) Hypodermal expression of the RAD-SMAD GFP reporter in wild type (A, E), <i>dbl-1(wk70)</i> (B, F), <i>lon-2(e678)</i> (C, G) and <i>tsp-21(jj77)</i> (D, H) worms at the L2 stage. The exposure time for all the GFP images was identical. (I) Quantification of the hypodermal RAD-SMAD GFP fluorescence intensity in various mutants compared with wild-type animals (set to 1). *** <i>p</i><0.0001, (unpaired two-tailed Student’s <i>t</i>-test). Error bars represent 95% confidence intervals for the normalized RAD-SMAD intensity.</p

TSP-21 is localized to the plasma membrane of multiple cell types, including known Sma/Mab signal-receiving cells.

<p>(A-R) Mid-stage embryo (A-C) or L1 larvae (D-L) or L4 larvae (M-R) showing confocal images of TSP-21::GFP (A, D, G, J, M, P), the corresponding DIC (B, E, H, K, N, Q) and merged images (C, F, I, L, O, R). TSP-21::GFP is localized to the plasma membrane of hypodermal and pharyngeal cells in embryos (A-C), pharyngeal cells (D-F), intestinal cells (G-I) and hypodermal cells (J-L) in L1 larvae, the developing gonad (M-O) and rectal epithelium (P-R) in L4 larvae. (S-Z) L1 larvae expressing both TSP-21::GFP and the M lineage specific reporter <i>hlh-8p</i>::<i>nls</i>::<i>rfp</i>. TSP-21::GFP is present in the M lineage from the 1-M stage through the 16-M stage. Some M lineage cells are out of the focal plane and not shown in panels U-V and Y-Z.</p

Suppression of the <i>sma-9(0)</i> M lineage defects by <i>tsp-21</i> mutations.

<p>Suppression of the <i>sma-9(0)</i> M lineage defects by <i>tsp-21</i> mutations.</p

Summary of the mutant alleles isolated in the <i>sma-9</i> suppressor screen.

<p>Suppression was determined based on the number of M lineage-derived coelomocytes (CCs). <i>sma-9(cc604)</i> hermaphrodites have 0 M lineage-derived CCs, while wild-type worms have 2.</p><p>ND: not determined</p><p>^a The worms scored are homozygous for <i>sma-9(cc604)</i> and homozygous for the suppressor indicated.</p><p>^b The worms scored are homozygous for <i>sma-9(cc604)</i> and heterozygous for the suppressor indicated.</p><p>^c Reported in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005221#pgen.1005221.ref020" target="_blank">20</a>].</p><p>^d Reported in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005221#pgen.1005221.ref021" target="_blank">21</a>].</p><p>^e Reported in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005221#pgen.1005221.ref022" target="_blank">22</a>].</p><p>^f Both <i>jj65</i> and <i>jj85</i> complemented <i>dbl-1(wk70)</i> and do not carry any molecular lesions in the <i>dbl-1</i> coding or 5’ and 3’ regulatory regions.</p><p>^g<i>jj58</i> might be a dominant <i>sma-9</i> suppressor and was not characterized in this study.</p><p>^h<i>jj68</i>, <i>jj80</i>, <i>jj81</i> and <i>jj84</i> were not characterized in this study due to their low degree of suppression of the <i>sma-9</i> M lineage phenotype.</p><p>ⁱ Predicted based on likely splicing defect.</p><p>^j The predicted truncation at the amino acid level was calculated assuming that the large deletion does not affect the stability of the <i>lon-2</i> message in <i>jj61</i> animals. The deletion also deletes the majority of the <i>aman-1</i> coding region, leaving only 338bp of the <i>aman-1</i> first exon to be present. The Susm phenotype is due to the loss of <i>lon-2</i> function because <i>jj61</i> failed to complement <i>lon-2(e678)</i> for the Susm phenotype.</p><p>^k Aberrant transcripts were confirmed by RT-PCR and reported in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005221#pgen.1005221.ref022" target="_blank">22</a>].</p><p>^l Corresponding genes were identified via a combination of linkage analysis (linkage to <i>cup-5(ar465)</i> III, <i>sma-9(cc604) X</i>), snip-SNP mapping [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005221#pgen.1005221.ref086" target="_blank">86</a>], and complementation tests based on the Susm phenotype, with the following alleles: <i>dbl-1(wk70)</i>, <i>sma-6(e1482)</i>, <i>daf-4(m63)</i>, <i>sma-2(e502)</i>, <i>sma-3(e491)</i>, <i>sma-4(e729)</i>, <i>lon-2(e678)</i>, and <i>lon-1(e185)</i>.</p><p>^m Corresponding genes were identified via WGS (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005221#sec015" target="_blank">Materials and methods</a>).</p><p>ⁿ Corresponding genes were identified via SNP-WGS (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005221#sec015" target="_blank">Materials and methods</a>).</p><p>^o In addition to carrying a mutation in <i>sma-4</i>, <i>jj70</i> appears to carry another <i>sma-9</i> suppressing mutation that maps to LG V and fails to complement <i>jj65</i> and <i>jj85</i>.</p><p>^p All molecular lesions were identified or confirmed by Sanger Sequencing.</p><p>^q A fragment containing 3kb of upstream sequences, the genomic coding region and 2kb of downstream sequences of <i>sma-6</i> rescued the Susm phenotype of <i>jj69</i>.</p><p>Summary of the mutant alleles isolated in the <i>sma-9</i> suppressor screen.</p