Craniosynostosis‐associated variants in the IL‐11R complex: new insights and questions

Skull growth involves the expansion of both the flat calvarial bones of the skull and the fibrous marginal zones, termed sutures, between them. This process depends on co‐ordinated proliferation of mesenchymal‐derived progenitor cells within the sutures, and their differentiation to osteoblasts which produce the bone matrix required to expand the size of the bony plates. Defects lead to premature closure of these sutures, termed craniosynostosis, resulting in heterogeneous head shape differences due to restricted growth of one or more sutures. The impact on the individual depends on how many and which sutures are affected and the severity of the effect. Several genetic loci are responsible, including a wide range of variants in the gene for the interleukin 11 receptor (IL11RA, OMIM#600939). Recent work from Kespohl and colleagues provides new insights into how some of these variants influence IL‐11R function; we discuss their influences on IL‐11R structure and IL‐11 function as a stimulus of osteoblast differentiation.


Introduction
Interleukin 11 (IL-11) is an IL-6 family cytokine.It signals by binding first to the IL-11 receptor (IL-11R) subunit, followed by sequential binding of the receptor-bound ligand to two molecules of the common IL-6 signal-transducing receptor, gp130, encoded by the IL6ST gene [1]; this initiates JAK/STAT phosphorylation [2].A wide range of variants in the gene for the interleukin 11 receptor (IL11RA, OMIM#600939) have been associated with craniosynostosis [3][4][5].Confirming that these most likely relate to loss of function, null mice for IL-11R also exhibit a partially penetrant craniosynostosis phenotype [3,6].Recent work from Birte Kespohl et al. [7] has provided new insights into how some of these variants influence IL-11R function.
A role for IL-11 signalling in craniosynostosis relates to its function as a stimulus of osteoblast differentiation.Skull growth involves proliferation of mesenchymal-derived progenitor cells within the calvarial sutures between the bony plates, and their differentiation to osteoblasts which deposit new bone matrix at the margins, thereby enlarging the bony plates.IL-11 has been shown to stimulate osteoblast differentiation by treatment of progenitors with recombinant protein in vitro [8,9], and this function was confirmed in vivo when increased bone formation was detected in a mouse model overexpressing IL-11 [10].Mice lacking the IL-11 specific receptor alpha subunit (IL-11RA) exhibit low rates of bone formation both on bone surfaces in the marrow environment [8] and on the outer surfaces of bone (the periosteum) leading to narrower bones in the appendicular skeleton [11].The effect of IL-11 on bone formation occurs through at least two mechanisms: by promoting commitment of pluripotent progenitors towards the osteoblast lineage while suppressing their differentiation to adipocytes [8] and by reducing production of the Wnt inhibitor sclerostin, a naturally occurring "brake" on bone formation produced by cells within the bone matrix termed osteocytes [12].

Multiple variants in IL-11 signalling associated with craniosynostosis
The multiple variants in both IL11RA and IL6ST associated with craniosynostosis point to the complexity of processes required for formation of the IL-11R signalling complex.To date, three potential mechanisms that impair IL-11R signalling have been associated with craniosynostosis: disruption of IL-11R near the IL-11 ligand binding site [3], disruption of gp130 function leading to cytokine-specific signalling defects [13][14][15], and disruption of IL-11R post-translational modifications required for receptor migration to the cell membrane [6,7].Given the complexity of the IL-11R signalling complex and the mechanisms required for all components to interact appropriately at the cell surface, there may be more.For example, variants in domain 1 of gp130 have the potential to disrupt IL-11 and IL-6 signalling, while leaving signalling by other IL-6 family members intact [1].
These mechanisms are not mutually exclusive since variants in either receptor may cause dual functional defects.For example, destabilising variants may alter processing and trafficking while also reducing assembly of the signalling complex.For example, molecular dynamics simulations showed that the IL-11R R296W variant destabilised domain 3 and increased flexibility of the domain 2/domain 3 interface, which is the location of ligand binding [16].Domain 3 also contains a highly conserved tryptophan-arginine ladder; this is a hotspot for craniosynostosis variants (Fig. 1), including W307R [17] and T306_S308dup [7] which both exhibit defects in receptor transport to the cell membrane.The tryptophan-arginine ladder also contains the WSXWS motif which is common to many type-I cytokine receptors and has been proposed to function as a stabiliser for complex formation [18].Similarly, variants in IL6ST likely lead to loss of IL-11 signalling by specific disruption of the gp130/IL-11R interface [13] or by altering gp130 conformational dynamics [15].
The first work to identify IL-11R variants associated with craniosynostosis provided some evidence of impaired ligand binding and STAT3 signalling [3].The R296W variant was studied in most detail, and no change in intracellular localisation or protein levels  was found by western blot at that time.More recent work showed that this variant of IL-11R transfected into HEK293 cells exhibited retention of IL-11R in the endoplasmic reticulum (ER) due to failure of transport to the cell membrane, and that this may have originated in defective post-translational glycosylation of the receptor [6].Defective glycosylation and trafficking have been studied in a similar manner and proposed for the recently described IL-11R T306_S308dup variant [7].Such post-translational modifications occur in the ER and are common to many transmembrane proteins.This includes N-linked glycosylation, which is subject to glycan-dependent quality control before the protein exits to the Golgi and is trafficked to the plasma membrane.It is striking that impaired Nlinked glycosylation of gp130 did not reduce its transport to the plasma membrane nor its signalling [19].Clearly, this is different for IL-11R.Post-translational modifications required to support receptor folding and transport may also include C-mannosylation, since Kespohl et al. [7] show that IL-11R is natively Cmannosylated.
The second IL-11R craniosynostosis-associated variant studied by Kespohl et al. [7], E364_V368del, showed no maturation or signalling defect.The authors suggest that E364_V368del, while present, is not causative of craniosynostosis in this patient.This recalls recent work in null mice for IL-11 ligand, which also exhibited no craniosynostosis, suggesting IL-11-independent functions of IL-11R [20].Such a suggestion is supported by the knowledge that although multiple human IL11RA variants have been associated with craniosynostosis, no genetic variants in the ligand (IL11) have been associated with this condition.However, IL-11 is clearly important for osteoblast differentiation, a second report using both a germline IL-11 ligand null mouse, and an osteoblastspecific knockout of IL-11 reported impaired osteoblast differentiation in both models, including elevated sclerostin and reduced Wnt signalling [21].However, neither presence or absence of craniosynostosis nor any effect on periosteal bone formation was reported.

Conclusions
Given the partial penetrance of the craniosynostosis phenotype in IL-11R null mice, and with the human IL11R and IL6ST variants [7,13], further regulatory mechanisms determine whether these variants (and any variation in IL11) result in a phenotype.This may include interactions with the very many other gene variants associated with craniosynostosis (such as TWIST1, FGFRs, and EPHB1) and with environmental and epigenetic factors.Further investigation will no doubt shed new light on these outstanding questions surrounding the roles of IL-11 signalling and the IL-11 signalling complex components in bone formation and craniosynostosis.

Fig. 1 .
Fig. 1.Domain structure of the IL-11R protein, showing variants associated with craniosynostosis to date.Highlighted with circles are those variants reported to exhibit impaired STAT3 phosphorylation (grey circles), impaired receptor transport to the cell membrane (open circles), and to retain biological activity (black circle).Variants in blue are within residues that comprise the arginine-tryptophan ladder, which is highlighted in domain 3 (D3) by white lines (arginines) and yellow lines (tryptophans).SP = signal protein, D1 = domain 1, D2 = domain 2. Structure drawn by the author with some amino acid locations sourced from ProteinPaint.