Development and validation of subject-specific pediatric multibody knee kinematic models with ligamentous constraints.

Computational knee models that replicate the joint motion are important tools to discern difficult-to-measure functional joint biomechanics. Numerous knee kinematic models of different complexity, with either generic or subject-specific anatomy, have been presented and used to predict three-dimensional tibiofemoral (TFJ) and patellofemoral (PFJ) joint kinematics of cadavers or healthy adults, but not pediatric populations. The aims of this study were: (i) to develop subject-specific TFJ and PFJ kinematic models, with TFJ models having either rigid or extensible ligament constraints, for eight healthy pediatric participants and (ii) to validate the estimated joint and ligament kinematics against in vivo kinematics measured from magnetic resonance imaging (MRI) at four TFJ flexion angles. Three different TFJ models were created from MRIs and used to solve the TFJ kinematics: (i) 5-rigid-link parallel mechanism with rigid surface contact and isometric anterior cruciate (ACL), posterior cruciate (PCL) and medial collateral (MCL) ligaments (ΔLnull), (ii) 6-link parallel mechanism with minimized ACL, PCL, MCL and lateral collateral ligament (LCL) length changes (ΔLmin) and (iii) 6-link parallel mechanism with prescribed ACL, PCL, MCL and LCL length variations (ΔLmatch). Each model's geometrical parameters were optimized using a Multiple Objective Particle Swarm algorithm. When compared to MRI-measured data, ΔLnull and ΔLmatch performed the best, with average root mean square errors below 6.93° and 4.23 mm for TFJ and PFJ angles and displacements, respectively, and below 2.01 mm for ligament lengths (<4.32% ligament strain). Therefore, within these error ranges, ΔLnull and ΔLmatch can be used to estimate three-dimensional pediatric TFJ, PFJ and ligament kinematics and can be incorporated into lower-limb models to estimate joint kinematics and kinetics during dynamic tasks.

Feasibility of using MRIs to create subject-specific parallel-mechanism joint models.

Musculoskeletal models typically use generic 2D models for the tibiofemoral (TFJ) and patellofemoral (PFJ) joints, with a hinge talocrural joint (TCJ), which are scaled to each subject׳s bone dimensions. Alternatively joints' measured kinematics in cadavers are well-predicted using 3D cadaver-specific models. These employ mechanisms constrained by the articulations of geometric objects fitted to the joint׳s surfaces. In this study, we developed TFJ, PFJ and TCJ mechanism-based models off MRIs for fourteen participants and compared the estimated kinematics with those from published studies modified to be consistent with mechanisms models and subject-specific anatomical landmarks. The models' parameters were estimated by fitting spheres to segmented articular cartilage surfaces, while ligament attachment points were selected from their bony attachment regions. Each participant׳s kinematics were estimated by ensuring no length changes in ligaments and constant distances between spheres' centres. Two parameters' optimizations were performed; both avoid singularities and one best matches the kinematic patterns off published studies. Sensitivity analysis determined which parameters the models were sensitive to. With both optimization methods, kinematics did not present singularities but correlation values were higher, exceeding 0.6, when matching the published studies. However, ranges of motion (ROM) were different between estimated and published studies. Across participants, models presented large parameter variation. Small variations were found between estimated- and optimized-parameters, and in the estimated-rotations and translations' means and ROM. Model results were sensitive to changes in distal tibia, talus and patella spheres' centres. These models can be implemented in subject-specific rigid-body musculoskeletal models to estimate joint moments and loads.

LMTK2-mediated phosphorylation regulates CFTR endocytosis in human airway epithelial cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-)-selective ion channel expressed in fluid-transporting epithelia. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser(737) in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl(-) secretion mediated by the wild-type and rescued ΔF508-CFTR. Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-Ser(737) mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl(-) channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl(-) channels and improve stability of pharmacologically rescued ΔF508-CFTR in patients with cystic fibrosis.

CFTR mutations altering CFTR fragmentation.

Most CF (cystic fibrosis) results from deletion of a phenylalanine (F508) in the CFTR {CF transmembrane-conductance regulator; ABCC7 [ABC (ATP-binding cassette) sub-family C member 7]} which causes ER (endoplasmic reticulum) degradation of the mutant. Using stably CFTR-expressing BHK (baby-hamster kidney) cell lines we demonstrated that wild-type CTFR and the F508delCFTR mutant are cleaved into differently sized N- and C-terminal-bearing fragments, with each hemi-CFTR carrying its nearest NBD (nucleotide-binding domain), reflecting differential cleavage through the central CFTR R-domain. Similar NBD1-bearing fragments are present in the natively expressing HBE (human bronchial epithelial) cell line. We also observe multiple smaller fragments of different sizes in BHK cells, particularly after F508del mutation (ladder pattern). Trapping wild-type CFTR in the ER did not generate a F508del fragmentation fingerprint. Fragments change their size/pattern again post-mutation at sites involved in CFTR's in vitro interaction with the pleiotropic protein kinase CK2 (S511A in NBD1). The F508del and S511A mutations generate different fragmentation fingerprints that are each unlike the wild-type; yet, both mutants generate new N-terminal-bearing CFTR fragments that are not observed with other CK2-related mutations (S511D, S422A/D and T1471A/D). We conclude that the F508delCFTR mutant is not degraded completely and there exists a relationship between CFTR's fragmentation fingerprint and the CFTR sequence through putative CK2-interactive sites that lie near F508.

Regulation of ENaC biogenesis by the stress response protein SERP1.

Cystic fibrosis lung disease is caused by reduced Cl(-) secretion along with enhanced Na(+) absorption, leading to reduced airway surface liquid and compromised mucociliary clearance. Therapeutic strategies have been developed to activate cystic fibrosis transmembrane conductance regulator (CFTR) or to overcome enhanced Na(+) absorption by the epithelial Na(+) channel (ENaC). In a split-ubiquitin-based two-hybrid screening, we identified stress-associated ER protein 1 (SERP1)/ribosome-associated membrane protein 4 as a novel interacting partner for the ENaC ß-subunit. SERP1 is induced during cell stress and interacts with the molecular chaperone calnexin, thus controlling early biogenesis of membrane proteins. ENaC activity was measured in the human airway epithelial cell lines H441 and A549 and in voltage clamp experiments with ENaC-overexpressing Xenopus oocytes. We found that expression of SERP1 strongly inhibits amiloride-sensitive Na(+) transport. SERP1 coimmunoprecipitated and colocalized with ßENaC in the endoplasmic reticulum, together with the chaperone calnexin. In contrast to the inhibitory effects on ENaC, SERP1 appears to promote expression of CFTR. Taken together, SERP1 is a novel cochaperone and regulator of ENaC expression.

Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity.

Previously, the pleiotropic "master kinase" casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.

Antagonistic regulation of cystic fibrosis transmembrane conductance regulator cell surface expression by protein kinases WNK4 and spleen tyrosine kinase.

Members of the WNK (with-no-lysine [K]) subfamily of protein kinases regulate various ion channels involved in sodium, potassium, and chloride homeostasis by either inducing their phosphorylation or regulating the number of channel proteins expressed at the cell surface. Here, we describe findings demonstrating that the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is also regulated by WNK4 in mammalian cells. This effect of WNK4 is independent of the presence of kinase and involves interaction with and inhibition of spleen tyrosine kinase (Syk), which phosphorylates Tyr512 in the first nucleotide-binding domain 1 (NBD1) of CFTR. Transfection of catalytically active Syk into CFTR-expressing baby hamster kidney cells reduces the cell surface expression of CFTR, whereas that of WNK4 promotes it. This is shown by biotinylation of cell surface proteins, immunofluorescence microscopy, and functional efflux assays. Mutation of Tyr512 to either glutamic acid or phenylalanine is sufficient to alter CFTR surface levels. In human airway epithelial cells, downregulation of endogenous Syk and WNK4 confirms their roles as physiologic regulators of CFTR surface expression. Together, our results show that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk perform an antagonistic role in this process.