Polycystin‐1 regulates cell proliferation and migration through AKT/mTORC2 pathway in a human craniosynostosis cell model

Abstract Craniosynostosis is the premature fusion of skull sutures and has a severe pathological impact on childrens’ life. Mechanical forces are capable of triggering biological responses in bone cells and regulate osteoblastogenesis in cranial sutures, leading to premature closure. The mechanosensitive proteins polycystin‐1 (PC1) and polycystin‐2 (PC2) have been documented to play an important role in craniofacial proliferation and development. Herein, we investigated the contribution of PC1 to the pathogenesis of non‐syndromic craniosynostosis and the associated molecular mechanisms. Protein expression of PC1 and PC2 was detected in bone fragments derived from craniosynostosis patients via immunohistochemistry. To explore the modulatory role of PC1 in primary cranial suture cells, we further abrogated the function of PC1 extracellular mechanosensing domain using a specific anti‐PC1 IgPKD1 antibody. Effect of IgPKD1 treatment was evaluated with cell proliferation and migration assays. Activation of PI3K/AKT/mTOR pathway components was further detected via Western blot in primary cranial suture cells following IgPKD1 treatment. PC1 and PC2 are expressed in human tissues of craniosynostosis. PC1 functional inhibition resulted in elevated proliferation and migration of primary cranial suture cells. PC1 inhibition also induced activation of AKT, exhibiting elevated phospho (p)‐AKT (Ser473) levels, but not 4EBP1 or p70S6K activation. Our findings indicate that PC1 may act as a mechanosensing molecule in cranial sutures by modulating osteoblastic cell proliferation and migration through the PC1/AKT/mTORC2 cascade with a potential impact on the development of non‐syndromic craniosynostosis.


| INTRODUC TI ON
Craniosynostosis refers to unsynchronized ossification of cranial sutures giving rise to both syndromic and non-syndromic subtypes. 1 The syndromic subtype is characterized by prematurely fused sutures and several morphological manifestations along with genetic abnormalities. Common symptoms include malformed skull shape, high levels of intracranial pressure, visual or respiratory deficiencies, and neurological dysfunction. It has been linked to various epidemiological factors such as multiple pregnancies, prematurity, birthweight and parents' age. 2,3 The non-syndromic craniosynostosis subtypes account for 70% of the cases and are mainly characterized by fused sutures. Several mutations have been detected in osteoblastogenic genes encoding the fibroblast growth factor receptors (FGFRs), homeobox protein MSX-2 (MSX2), ephrin-B (EFNB), twist-related protein 1 (TWIST1) and runt-related transcription factor 2 (Runx2). However, the underlying molecular mechanisms remain largely unknown. 4 Dysfunctional mechanical inputs in the microenvironment contribute to the pathogenesis of craniosynostosis. [5][6][7] Masticatory forces can induce premature sagittal suture closure in osteopetrotic mice, yet fusion of the internasal suture occurs in mice following soft diet. 5,7 The presence of extrinsic pathological forces is a contributory factor to premature fusion. Non-syndromic craniosynostosis has been linked to low foetal station, multiple births and malpresentation. Notably, increased intrauterine forces belong to the common aetiology. 6 Transient receptor potential channels (TRPs) mediate sensory signals and consist of more than 30 cation channels, which are further subdivided into six subfamilies in mammals: the canonical (TRPC), the vanilloid (TRPV), the melastatin (TRPM), the polycystin (TRPP), the mucolipin (TRPML) and the ankyrin (TRPA) subfamilies. 8 TRP channels are activated by external or intrinsic stimuli and regulate various physiological and pathological functions. Mutations in TRP genes are causative agents in the pathogenesis of TRP channelopathies. 9 All TRPs seem to have six transmembrane domains, which assemble as homo-or hetero-tetramers within the channel. 10 Various intracellular and extracellular factors, such as chemical and osmotic stress, trigger the activation of TRPs. 11 At the extracellular level, TRPs sense signals including chemical, osmotic and mechanical stress. 11 In several types of cells, they are involved in thermosensation and taste reception. 12,13 The abundance of intracellular Ca 2+ stores is sensed by TRPs and thus stimulates signal transduction pathways for the restoration of Ca 2+ balance. TRPs also contribute to the changes and balance of the concentration of free cytosolic Ca 2+ . 14 Being located intracellularly or at the plasma membrane, TRPs are also involved in entry and release pathways of Ca 2+ from cell organelles facilitating its transport. 10 The mechanosensory molecules and TRP channels, PC1 and PC2, have been implicated in flow mechanosensation, brain injury, skeletal development and osteoblast differentiation. [15][16][17] Polycystins are expressed in human tissues, including kidneys, blood vessels, pancreas, liver, bone and skull. Being localized at the primary cilium, at the plasma membrane and at the endoplasmic reticulum (ER), they interact with other molecules, connecting the extracellular matrix with the cytoskeleton and thus igniting intracellular signalling pathways. 18 The intracellular PC1 C-terminal tail (CT) has been demonstrated to interact and activate several signal transduction pathways including Janus activating kinase (JAK)-signal transducer and activator of transcription (STAT), the mechanistic target of rapamycin (mTOR), Wnt, the activator protein-1 (AP-1) and the calcineurinnuclear factor of activated T-cell (NFAT) pathways. [18][19][20] Polycystin-1-deficient mice subjected to midpalatal suture expansion and presented craniofacial deformities at the skull base and in craniofacial sutures, a finding which could not be related to signalling mechanisms, though. 16 Moreover, mutant mice with a conditional deletion of the polycystic kidney disease 2 (PKD2) gene, which encodes for PC2, in neural crest-derived cells exhibited dysfunctional skull development, such as mechanical trauma, fractured molar roots, distorted incisors, alveolar bone loss and compressed temporomandibular joints, in addition to abnormal skull shapes. 17 There is also accumulating evidence that mTOR signalling is essential for normal skeletal growth. 21,22 Discovered in the early 1990s, mTOR is involved in the regulation of essential cell processes. 23-25 A dysfunctional mTOR signalling has been related to various pathogeneses such as cancer and neurodegenerative diseases. 26,27 More specifically, osteogenesis and craniosynostosis have both been correlated with mTOR signalling. 21,22 Proliferation and inactivity of stem cells in the adult forebrain are also regulated by mTOR. 28 The upstream effectors of mTOR, phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) are key regulators of the differentiation of various cell types including chondrocytes, osteoblasts, myoblasts and adipocytes. 29 PI3K acts as a catalyst and results in the production of phosphatidylinositol-3,4,5-trisphosphate, activating various signalling components of gene expression and regulators of cell survival. 30 PI3K is an osteoblast differentiation regulator, interacting with local signalling factors 31-33 and the tissue-specific Runx2. 34 PI3K/AKT/mTOR pathway is also involved in the control of the pluripotent stem cells. 35,36 Since PC1 has previously been shown to induce mTOR signalling and regulate mTOR pathway components activity, 37,38 we proceeded to investigate the potential implication of PC1/PI3K/AKT/ mTOR signalling network in craniosynostosis and its effect on the cell properties of primary cranial suture cells.

| Tissue samples
The study included 17 suture bone fragments of non-syndromic crani-

| Reagents and antibodies
Cell culture media and all tissue culture reagents were obtained from Gibco (ThermoFisher Scientific) and Biosera. For immunohistochemistry, the following reagents were used: Dako Real Envision Detection System, peroxidase/DAB1, rabbit/mouse (Dako). The following primary antibodies were employed for Western blot analysis:

| Primary suture cranial cell cultures
Extracts of human suture tissue from 5 patients (P) with craniosynostosis (3 with trigonocephaly and 2 with dolichocephaly) were isolated by collagenase digestion, and cranial suture cells were cultured according to the methods by Coussens et al. 39 In brief, the human suture tissue samples were dissected and minced into 1-mm bone fragments and incubated in 0.25% collagenase for 2 h at 37°C. Samples then were centrifuged, and the supernatant was removed. Samples were then extensively washed with PBS and plated at 5 bone fragments per well, in both 6-well and 12-well plates. Cells were cultured in minimal medium in a humidified atmosphere containing 5% CO 2 kept at 37°C. Minimal medium consisted of aMEM (Gibco, ThermoFisher Scientific), low glucose, supplemented with L-glutamine, 10% foetal bovine serum (FBS) (Gibco, ThermoFisher Scientific) and 1% antibiotics (penicillin 100 IU/ml, streptomycin 100 μg/ml) (Gibco, ThermoFisher Scientific). Upon confluency, cells were plated in T25 flasks and labelled P1. Medium was changed every 2 days. Cells were passaged to P4 to obtain sufficient amount of cells. All experiments were carried out with cells from the first to the fourth passage after being checked for their osteoblastic characteristics.

| Immunohistochemistry
Paraffin-embedded tissue specimens were examined by immu-

| Cell proliferation assay
Primary cranial suture cells were cultured in 96-well culture plates.

| Cell migration assay
Primary cranial suture cells were cultured in 6-well culture plates.

| Statistical and image analysis
Statistical analyses were conducted with the SPSS 23.0 and Microsoft Excel software packages. Correlation tests were carried out. All experiments were performed at least three times, and representative results of one experiment are shown. The data are presented as mean ± SE for the number of experiments indicated and analysed by Student's t-test. All statistical tests were two-sided. pvalues < 0.05 were regarded as statistically significant. The Image J software was used for densitometry quantification analysis.

| Detection and localization of polycystins in human craniosynostosis samples
We initially evaluated polycystins' localization and expression in suture tissue samples by immunohistochemistry. PC1 localization was detected in the cytoplasm of osteoblasts and osteocytes in both craniosynostosis subtypes ( Figure 1A,B). PC1 expression in osteoblasts ranged from 0 to 75% (mean value 28%), whereas in osteocytes ranged from 0 to 75% (mean value 33%). The overall H-score of PC1 ranged from 0 to 225 in osteoblasts and osteocytes with a median up to 25.
Polycystin-2 localization was also observed in osteoblasts and osteocytes with mostly cytoplasmic expression. PC2 expression ranged from 0 to 100% (mean value 43%), whereas in osteocytes, PC2 ranged from 0 to 75% (mean value 34%). The overall PC2 Hscore in osteoblasts ranged from 0 to 300 with a median of 50. In osteocytes, the median of H-score was 38, ranging from 0 to 300.  Figure 1C). Upon IgPKD1 treatment, trigonocephaly cranial cells exhibited higher proliferation (p < 0.05) compared to untreated cells ( Figure 2A). Dolichocephaly cells also exhibited increased proliferation (p < 0.001) upon PC1 inhibition compared to controls ( Figure 2D). The effects of PC1 on cell migration were further studied using the wound healing assay ( Figure 2B,E). Trigonocephaly cranial suture cells showed increased migration upon IgPKD1 treatment compared to untreated cells (p < 0.001) ( Figure 2B,C) whereas dolichocephaly cranial suture cells did not demonstrate such a prominent migratory potential upon IgPKD1 treatment compared to untreated cells (p = ns) ( Figure 2E,F).

| PC1 inhibition induces activation of mTOR pathway components in human primary cranial suture cells
Knowing the association between PC1 and mTOR pathways in other pathophysiologies, 37

| PC1 inhibition induces specific activation of AKT/mTORC2 signalling in craniosynostosis
The mTOR complex consists of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 has 4EBP1 and p70S6K among its downstream effectors, while mTORC2 is reciprocally activated with AKT. 43  Following the original detection of polycystins in tissue samples, we proceeded to investigate the functional role of PC1 in the cellular processes of human primary cranial suture cells. We focused on PC1 because of its main role in mechanosensation, cell-to-cell adhesion and cell-matrix interactions. 51 We have used a well-characterized antibody (IgPKD1) that binds specifically to the extracellular domain of PC1 and blocks its mechanosensing ability. 20,[40][41][42]52 In this way, PC1 inhibition was shown to promote certain features such as increasing proliferation and migration of cranial cells derived from trigonocephaly and dolichocephaly suture tissues, a finding which was in concert with previous studies, where absent or dysfunctional polycystin exhibited increased proliferation and migration. 40,42,52 Polycystin-1 has been implicated in osteogenesis 53 and in craniofacial development of mouse models. 16,17 However, its precise role in signalling is not yet fully elucidated in cranial sutures and craniosynostosis. Thus, our next experiments focused on identifying novel interactions in signal transduction pathways. Having identified elevated migration and cell proliferation, we focused on the mTOR signalling pathway as it is connected to PC1 activity and biology of the skeletal tissue. 21,34,37,38,41,42,[54][55][56][57] Mechanistic target of rapamycin is implicated in osteogenesis holding a significant role. 54 42 In agreement with the above data, we further examined this connection of PC1 and mTOR components in human cranial suture cells. An induction of p-AKT levels in human cranial suture cells upon PC1 inhibition that was specific to craniosynostotic cells was detected, suggesting its implication in the disease phenotype. To further strengthen this observation, we treated cells with a PI3K inhibitor of the PI3K/AKT/ mTOR pathway. In this case, p-AKT and p-mTOR were abrogated, an effect that was then reversed when cells were treated with both PI3K and IgPKD1 inhibitors, suggesting that PC1 inhibition can relate to mTOR pathway activation, thus cell migration and proliferation.
Furthermore, we attempted to investigate PTEN expression, which is involved in PI3K/mTOR pathway and is implicated in craniofacial morphogenesis in mice. 61 We

| CON CLUS ION
In summary, our research highlights the role of PC1 as a regulator of cell proliferation and migration and its interaction with mTOR signalling in human cranial cells. Given that there is a lack of prior research concerning the role of polycystins in craniosynostosis, the present study is one of the first steps towards understanding the function of polycystins in the pathophysiology of craniosynostosis in human conditions. Future experiments should focus on the mechanism through which PC1 promotes or inhibits cell proliferation and migration, and the molecular details of the interaction between PC1 and mTOR. Moreover, future studies on polycystins and craniosynostosis should explore whether polycystins are associated with any other signalling pathways related to osteogenesis. All the above will contribute to the identification of new prognostic markers in non-syndromic craniosynostosis as well as to elucidating the key role of PC1 in a new therapeutic scheme against craniosynostosis at a diagnosis level.
Studies on the mechanobiology of craniosynostosis and the respective effect of forces in cranial formation may reveal that craniosynostosis is, in part, a bone condition where mechanical stress and pressure during pregnancy or post-birth play a crucial role.
Such knowledge may advance our understanding of craniosynostosis pathobiology and reveal novel therapeutic targets, presumably using polycystin as a new tool against the development of craniosynostosis.

ACK N OWLED G EM ENTS
The IgPKD1 antibody was a generous gift from O. Ibraghimov-Beskrovnaya and H. Husson (Genzyme Co., Boston, MA). Studies F I G U R E 4 Effect of combined IgPKD1 and PI3K inhibitor treatment on mTOR signalling pathway in Trigonocephaly (T) and Dolichocephaly (D) cranial cells. (A and B) Western blot analysis and quantitative data from cell lysates of T cranial cells using p-mTOR, p-AKT and p-4EBP1 antibodies. (C and D) Western blot analysis and quantitative data from cell lysates of D cranial cells using p-mTOR, p-AKT and p-4EBP1 antibodies. Actin was used as a protein loading control. Representative Western blots of each cell line are presented. All data were analysed by t-test and represent the mean ± SD. The experiments have been performed in triplicate (Student's t-test, *p < 0.05, **p < 0.01, ***p < 0.001)

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.