Insights into KIF11 pathogenesis in microcephaly-lymphedema-chorioretinopathy syndrome from a lymphatic perspective
Kazim Ogmen, Sara E. Dobbins, Rose Yinghan Behncke, Ines Martinez-Corral, Ryan C.S. Brown, Michelle Meier, Sascha Ulferts, Nils Rouven Hansmeier, Ege Sackey, Ahlam Alqahtani, Christina Karapouliou, Dionysios Grigoriadis, Juan C. Del Rey Jimenez, Michael Oberlin, Denise Williams, Arzu Ekici, Kadri Karaer, Steve Jeffery, Peter Mortimer, Kristiana Gordon, Kazuhide S. Okuda, Benjamin M. Hogan, Taija Mäkinen, René Hägerling, Sahar Mansour, Silvia Martin-Almedina, Pia Ostergaard
Kazim Ogmen, Sara E. Dobbins, Rose Yinghan Behncke, Ines Martinez-Corral, Ryan C.S. Brown, Michelle Meier, Sascha Ulferts, Nils Rouven Hansmeier, Ege Sackey, Ahlam Alqahtani, Christina Karapouliou, Dionysios Grigoriadis, Juan C. Del Rey Jimenez, Michael Oberlin, Denise Williams, Arzu Ekici, Kadri Karaer, Steve Jeffery, Peter Mortimer, Kristiana Gordon, Kazuhide S. Okuda, Benjamin M. Hogan, Taija Mäkinen, René Hägerling, Sahar Mansour, Silvia Martin-Almedina, Pia Ostergaard
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Research Article Clinical Research Genetics Vascular biology

Insights into KIF11 pathogenesis in microcephaly-lymphedema-chorioretinopathy syndrome from a lymphatic perspective

Abstract

Pathogenic variants in kinesin KIF11 underlie microcephaly-lymphedema-chorioretinopathy (MLC) syndrome. Although well known for regulating spindle dynamics ensuring successful cell division, the association of KIF11 (encoding EG5) with development of the lymphatic system and how KIF11 pathogenic variants lead to lymphatic dysfunction and lymphedema remain unknown. Using patient-derived lymphoblastoid cells, we demonstrated that patients with MLC carrying pathogenic stop-gain variants in KIF11 have reduced mRNA and protein levels. Lymphoscintigraphy showed reduced tracer absorption, and intestinal lymphangiectasia was detected in one patient, pointing to impairment of lymphatic function caused by KIF11 haploinsufficiency. We revealed that KIF11 is expressed in early human and mouse development with the lymphatic markers VEGFR3, podoplanin, and PROX1. In zebrafish, single-cell RNA-Seq identified kif11 specifically expressed in endothelial precursors. In human lymphatic endothelial cells, EG5 inhibition with ispinesib reduced VEGFC-driven AKT phosphorylation, migration, and spheroid sprouting. KIF11 knockdown reduced PROX1 and VEGFR3 expression, providing for the first time to our knowledge a link between KIF11 and drivers of lymphangiogenesis and lymphatic identity.

Authors

Kazim Ogmen, Sara E. Dobbins, Rose Yinghan Behncke, Ines Martinez-Corral, Ryan C.S. Brown, Michelle Meier, Sascha Ulferts, Nils Rouven Hansmeier, Ege Sackey, Ahlam Alqahtani, Christina Karapouliou, Dionysios Grigoriadis, Juan C. Del Rey Jimenez, Michael Oberlin, Denise Williams, Arzu Ekici, Kadri Karaer, Steve Jeffery, Peter Mortimer, Kristiana Gordon, Kazuhide S. Okuda, Benjamin M. Hogan, Taija Mäkinen, René Hägerling, Sahar Mansour, Silvia Martin-Almedina, Pia Ostergaard

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Figure 8

KIF11 deficiency alters MAPK and AKT signaling pathways and reduces expression of lymphatic cell identity and cell-cell junction markers.

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KIF11 deficiency alters MAPK and AKT signaling pathways and reduces exp...
(A) Human phospho-kinase array showing changes in phosphorylation levels of 43 kinases. Dots have been magnified for selected kinases with decreased (in blue) or increased (in red) phosphorylation. (B) Network pathway analysis using STRING and Cytoscape reveals known interactions and the directionality of the phosphorylation change in siRNA KIF11-treated cells (blue, decreased; red, increased). (C) Effect of ispinesib treatment (50 nM) on AKT and MAPK phosphorylation in LECs over time after stimulation with VEGFC (100 ng/mL). DMSO (vehicle) as control. Phosphorylation was analyzed with anti-pAKT S473 and anti-p44/42 MAPK Thr202/Tyr204 (upper panels) and levels of AKT and MAPK protein expression with anti-AKT and MAPK antibodies (lower panels). Molecular mass markers (in kDa) are indicated on the left. Blots are quantified on the right. *P < 0.05, **P < 0.01, ns, nonsignificant. Two-tailed unpaired Student’s t test. (D) FLT4 (VEGFR3), PROX1, PDPN, FOXC2, LYVE1, TJP1 (ZO-1), CLDN5, and CDH5 gene expression related to GAPDH in LECs treated with siRNA 6 KIF11 for 48 and 72 hours analyzed by qPCR (n = 3–4 experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ns, nonsignificant. Two-tailed unpaired Student’s t test. (E) Western blot analysis and quantification of PROX1 and VEGFR3 expression in LECs treated with siRNA KIF11 for 48 and 72 hours. GAPDH was used as control. **P < 0.01, ***P < 0.001, ns, nonsignificant. Two-tailed unpaired Student’s t test. (C–E) Bars represent mean relative expression ± SEM. (C and E) One representative image is shown from n = 3–4 experiments.