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The focal adhesion kinase inhibitor, PF-562,271, is currently in clinical development for cancer, however it is not known how PF-562,271 affects T cell function. Here, we demonstrate inhibitory effects of PF-562,271 on the activation of primary human and mouse T cells. PF-562,271 inhibits T cell receptor signaling-induced T cell adhesion to intercellular adhesion molecule-1 and T cell interactions with antigen-presenting cells. An additional focal adhesion kinase inhibitor, PF-573,228, and genetic depletion of focal adhesion kinase also impair T cell conjugation with antigen-presenting cells. PF-562,271 blocks phosphorylation of the signaling molecules zeta chain associate protein of 70 kDa, linker of activated T cells, and extracellular signal-regulated kinase, and impairs T cell proliferation. The effects observed on T cell proliferation cannot solely be attributed to focal adhesion kinase inhibition, as genetic depletion did not alter proliferation. The effect of PF-562,271 on T cell proliferation is not rescued when proximal T cell receptor signaling is bypassed by stimulation with phorbol-12-myristate-13-acetate and ionomycin. Taken together, our findings demonstrate that focal adhesion kinase regulates integrin-mediated T cell adhesion following T cell receptor activation. Moreover, our findings suggest that PF-562,271 may have immunomodulatory effects that could impact its therapeutic applications.

Progenitor B cells reside in complex bone marrow (BM) microenvironments where they receive signals for growth and maturation. We reported previously that the CXCL12-focal adhesion kinase (FAK)-VLA4 pathway plays an important role in progenitor B cell adhesion and migration. In this study, we have conditionally targeted in B cells FAK, and found that the numbers of progenitor pro-B, pre-B, and immature B cells are reduced by 30–40% in B cell-specific FAK knockout mice. When cultured in methylcellulose with IL-7 ± CXCL12, Fak-deleted pro-B cells yield significantly fewer cells and colonies. Using in situ quantitative imaging cytometry, we establish that in longitudinal femoral BM sections, pro-B cells are preferentially localized in close proximity to the endosteum of the metaphyses and the diaphysis. Fak deletion disrupts the nonrandom distribution of pro-B cells and induces the mobilization of pro-B cells to the periphery in vivo. These effects of Fak deletion on pro-B cell mobilization and localization in BM are amplified under inflammatory stress, that is, after immunization with nitrophenol-conjugated chicken γ-globulin in alum. Collectively, these studies suggest the importance of FAK in regulating pro-B cell homeostasis and maintenance of their spatial distribution in BM niches.

The formation of the myelin sheath is a crucial step during development since it enables fast and efficient propagation of signals within the limited space of the mammalian central nervous system (CNS). During the process of myelination, oligodendrocytes actively interact with the extracellular matrix (ECM), and these interactions are considered crucial for proper and timely completion of the myelin sheath. However, the exact regulatory circuits involved in the signaling events that occur between the ECM and oligodendrocytes are currently not fully understood. In the present study we, therefore, investigated the role of a known integrator of cell-ECM signaling, namely focal adhesion kinase (FAK), in CNS myelination via the use of conditional (oligodendrocyte-specific) and inducible FAK knock-out mice (Fakflox/flox:PLP/CreERT mice). When inducing FAK knock-out just prior to and during active myelination of the optic nerve, we observed a significant reduction in the number of myelinated fibers at postnatal day 14. In addition, our data revealed a decreased number of primary processes extending from oligodendrocyte cell bodies at this postnatal age and upon induction of FAK knock-out. In contrast, myelination appeared normal at postnatal day 28. Thus, our data suggest that FAK controls the efficiency and timing of CNS myelination during its initial stages by, at least in part, regulating oligodendrocyte process outgrowth and/or remodeling.

The adult, virgin mammary gland is a highly organized branched ductal network comprising two major cell types: myoepithelial and luminal epithelial cells. To study the role and mechanism of focal adhesion kinase (FAK)-mediated signaling in mammary gland development and differentiation, we used a conditional Fak-knockout mammary epithelial cell (MEC) transplantation model. Conditional Cre recombinase (Cre)-mediated Fak deletion in primary cultured MECs isolated from FAK(lox/lox)/Rosa26Cre-ERT2 donor mice caused loss of FAK in all mammary cells. Transplantation of Fak-knockout MECs in a cleared mammary fat pad of immune-deficient recipient mice resulted in development of new but dilated virgin ducts with a disrupted myoepithelial and luminal epithelial cell multilayer and aberrant ductal morphogenesis during pregnancy. In the absence of FAK, MECs spread poorly, showed enhanced Rho kinase (ROCK)-mediated cytoskeletal contractility, and failed to respond to receptor-mediated cytoskeletal remodeling. Likewise, FAK deficiency fully inhibited branching morphogenesis of mammary gland organoids in a ROCK-dependent manner. Altogether these data suggest a model in which FAK coordinates contractile forces in MECs to maintain the bilayered cellular organization of myoepithelial and luminal epithelial cells in ducts, thus allowing proper mammary gland development and function.

Migration and proliferation of cardiac fibroblasts (CFs) play an important role in the myocardial remodeling process. While many factors have been identified that regulate CF growth and migration, less is known about the signaling mechanisms involved in these processes. Here, we utilized Cre-LoxP technology to obtain focal adhesion kinase (FAK)-deficient adult mouse CFs and studied how FAK functioned in modulating cell adhesion, proliferation, and migration of these cells. Treatment of FAKflox/floxCFs with Ad/Cre virus caused over 70% reduction of FAK protein levels within a cell population. FAK-deficient CFs showed no changes in focal adhesions, cell morphology, or protein expression levels of vinculin, talin, or paxillin; proline-rich tyrosine kinase 2 (Pyk2) expression and activity were increased. Knockdown of FAK protein in CFs increased PDGF-BB-induced proliferation, while it reduced PDGF-BB-induced migration. Adhesion to fibronectin was not altered. To distinguish between the function of FAK and Pyk2, FAK function was inhibited via adenoviral-mediated overexpression of the natural FAK inhibitor FAK-related nonkinase (FRNK). Ad/FRNK had no effect on Pyk2 expression, inhibited the PDGF-BB-induced migration, but did not change the PDGF-BB-induced proliferation. FAK deficiency had only modest effects on increasing PDGF-BB activation of p38 and JNK MAPKs, with no alteration in the ERK response vs. control cells. These results demonstrate that FAK is required for the PDGF-BB-induced migratory response of adult mouse CFs and suggest that FAK could play an essential role in the wound-healing response that occurs in numerous cardiac pathologies.

The heart is the first organ to form, and its development involves an intricate and complex series of events that must occur in a coordinated spatial and temporal fashion (44, 60, 75). The heart develops from bilaterally symmetric cardiogenic primordia that migrate and fuse at the embryonic midline to form a functional primitive heart tube that subsequently undergoes rightward looping to orient the atrial and ventricular chambers and properly align the outflow tract (OFT). As the chambers mature, the trabecular layer within the ventricles is elaborated, endocardial cushions form and fuse, and the chamber walls grow and thicken as the cardiomyocytes continue to proliferate and differentiate. A critical event in cardiac development related to human congenital heart defects (CHD) is septal morphogenesis, which commences during midgestation. The single ventricle becomes septated by a process involving fusion of the muscular interventricular septum (IVS) with the endocardial cushions, and further septation and valve formation continues until birth to ensure unidirectional flow of blood. Since multiple cell types, including those derived from the myocardium, endocardium, epicardium, and neural crest, contribute to the complex process of valvuloseptal morphogenesis, it is not surprising that CHD associated with aberrant septation of the OFT are quite common. Indeed, CHD afflict nearly 1% of newborn infants each year, and defective valvuloseptal morphogenesis is the leading cause of preterm mortality in the United States (22, 23, 53, 59). Thus, understanding the precise mechanisms underlying cardiac morphogenesis will aid in the identification of new therapies to target a multitude of congenital diseases (48). Both forward and reverse genetic studies with Drosophila melanogaster, zebrafish, Xenopus laevis, and mice have been instrumental in determining the transcription factors required for vertebrate heart specification, patterning, and differentiation (42). Included in this growing list of key cardiac specification/differentiation transcription factors are Nk factors (cardioblast specification), MEF2 and GATA factors (cardiomyocyte differentiation), d- and e-Hand (right and left ventricle formation, respectively), and TBX factors (ventricular septation) (42). In spite of this wealth of information regarding the transcription factors that coordinate cardiac morphogenesis, the signaling mechanisms that regulate heart formation are only just beginning to be elucidated. Of note, fibroblast growth factor (FGF) signaling regulates mesodermal differentiation into cardiac primordia, BMPs (members of the transforming growth factor β2 [TGF-β2] superfamily) regulate cardioblast specification, cushion formation, and OFT septation, and the neuregulin growth factors ErbB2 and ErbB4 are required for the development of trabeculae (44). Genetic evidence indicates that the integrin class of fibronectin-binding adhesion receptors (α5β1 and others) can also regulate both the form and function of the heart (8, 21, 55, 56, 58, 66, 74). Integrin ligation drives the recruitment of a number of structural and signaling molecules to the ventral plasma membrane collectively termed a focal adhesion, which serves to link the force-generating actin cytoskeleton inside the cell to the extracellular matrix (ECM) and to coordinate the activation of downstream signaling pathways (28). The nonreceptor tyrosine kinase focal adhesion kinase (FAK) is strongly activated by both integrins and growth factors and is a likely candidate to integrate downstream signals from these diverse pathways during growth and development (50). FAK is expressed at relatively high levels in the mouse mesoderm at embryonic day 7.5 (E7.5) and continues to be expressed in the heart and several other tissues throughout adulthood (15). Germ line deletion of FAK resulted in mesodermal defects and embryonic lethality between E8.5 and E10. The fak−/− embryos showed a phenotype similar to that observed for both fibronectin- and α5-null mice (30, 74). Although a rudimentary, nonbeating heart was apparent in some of these embryos, serial sectioning through fak−/− hearts revealed a lack of separate mesocardial and endocardial layers, which is indicative of defective cardiomyocyte maturation (15). Interestingly, germ line deletion of the FAK binding partners paxillin and Crk-associated substrate (CAS) also led to embryonic lethality associated with similar cardiac defects (19, 25). Paxillin and CAS are both adapter proteins that upon phosphorylation by FAK recruit additional signaling molecules to the focal adhesion complex and cause subsequent activation of downstream mitogen-activated protein kinase and GTPase-mediated signaling cascades (51). Taken together, these studies indicate that modulation of focal adhesion-dependent signals likely plays an important role in cardiac development and/or function. In order to study the time- and tissue-dependent requirements for FAK in mouse development, Beggs et al. recently generated and characterized a mouse line (fakflox/flox) in which exon 20 of the FAK gene (encoding the ATP binding domain) is flanked by loxP sites (3). We recently used this line to conditionally delete FAK from adult cardiomyocytes by use of a well-characterized mlc2vCre line. Somewhat surprisingly, we found that FAK is dispensable for basal cardiac function and myocyte viability. However, FAK was required for appropriate age- and pressure overload-induced compensatory myocyte hypertrophy. Indeed, mice with myocyte-restricted depletion of FAK had a blunted hypertrophic response that manifested in compromised heart function but not premature death (12). Herein, to determine whether FAK might play a direct role in cardiac morphogenesis, we generated a mouse with embryonic FAK deficiency in nkx2-5-expressing cells by use of a recently described Cre knock-in mouse line (46). We found that embryonic deletion of FAK in the nkx2-5-expressing cells results in perinatal lethality associated with a profound subaortic ventricular septal defect (VSD) and an abnormal OFT alignment. FAK inactivation does not affect myocyte growth or survival but regulates myocyte migration, a function that may play a role in the proper fusion of OFT cushion tissue with the muscular IVS.

Integrins link the inside of a cell with its outside environment and in doing so regulate a wide variety of cell behaviors. Integrins are well known for their roles in angiogenesis and cell migration but their functions in bone formation are less clear. The majority of integrin signaling proceeds through focal adhesion kinase (FAK), an essential component of the focal adhesion complex. We generated transgenic mice in which FAK was deleted in osteoblasts and uncovered a previously unknown role in osteoblast differentiation associated with bone healing. FAK mutant cells migrated to the site of skeletal injury and angiogenesis was unaffected yet the transgenic mice still exhibited numerous defects in reparative bone formation. Osteoblast differentiation itself was unperturbed by the loss of FAK, whereas the attachment of osteoclasts to the bone matrix was disrupted in vivo. We postulate that defective bi-directional integrin signaling affects the organization of the collagen matrix. Finally, we present a compensatory candidate molecule, Pyk2, which localized to the focal adhesions in osteoblasts that were lacking FAK.

In response to alphabeta1 integrin signaling, transducers such as focal adhesion kinase (FAK) become activated, relaying to specific machineries and triggering distinct cellular responses. By conditionally ablating Fak in skin epidermis and culturing Fak-null keratinocytes, we show that FAK is dispensable for epidermal adhesion and basement membrane assembly, both of which require alphabeta1 integrins. FAK is also dispensible for proliferation/survival in enriched medium. In contrast, FAK functions downstream of alphabeta1 integrin in regulating cytoskeletal dynamics and orchestrating polarized keratinocyte migration out of epidermal explants. Fak-null keratinocytes display an aberrant actin cytoskeleton, which is tightly associated with robust, peripheral focal adhesions and microtubules. We find that without FAK, Src, p190RhoGAP, and PKL-PIX-PAK, localization and/or activation at focal adhesions are impaired, leading to elevated Rho activity, phosphorylation of myosin light chain kinase, and enhanced tensile stress fibers. We show that, together, these FAK-dependent activities are critical to control the turnover of focal adhesions, which is perturbed in the absence of FAK.

Macrophages are a key component of the innate immune system. In this study, we investigate how focal adhesion kinase (FAK) and the related kinase Pyk2 integrate adhesion signaling and growth factor receptor signaling to regulate diverse macrophage functions. Primary bone marrow macrophages isolated from mice in which FAK is conditionally deleted from cells of the myeloid lineage exhibited elevated protrusive activity, altered adhesion dynamics, impaired chemotaxis, elevated basal Rac1 activity, and a marked inability to form stable lamellipodia necessary for directional locomotion. The contribution of FAK to macrophage function in vitro was substantiated in vivo by the finding that recruitment of monocytes to sites of inflammation was impaired in the absence of FAK. Decreased Pyk2 expression in primary macrophages also resulted in a diminution of invasive capacity. However, the combined loss of FAK and Pyk2 had no greater effect than the loss of either molecule alone, indicating that both kinases function within the same pathway to promote invasion.

Morphogenesis of a vascular network requires dynamic vessel growth and regression. To investigate the cellular mechanism underlying this process, we deleted focal adhesion kinase (FAK), a key signaling mediator, in endothelial cells (ECs) using Tie2-Cre mice. Targeted FAK depletion occurred efficiently early in development, where mutants exhibited a distinctive and irregular vasculature, resulting in hemorrhage and lethality between embryonic day (e) 10.5 and 11.5. Capillaries and intercapillary spaces in yolk sacs were dilated before any other detectable abnormalities at e9.5, and explants demonstrate that the defects resulted from the loss of FAK and not from organ failure. Time-lapse microscopy monitoring EC behavior during vascular formation in explants revealed no apparent decrease in proliferation or migration but revealed increases in cell retraction and death leading to reduced vessel growth and increased vessel regression. Consistent with this phenotype, ECs derived from mutant embryos exhibited aberrant lamellipodial extensions, altered actin cytoskeleton, and nonpolarized cell movement. This study reveals that FAK is crucial for vascular morphogenesis and the regulation of EC survival and morphology.

Focal adhesion kinase (FAK) is a critical component in transducing signals downstream of both integrins and growth factor receptors. To determine how the loss of FAK affects the epidermis in vivo, we have generated a mouse model with a keratinocyte-restricted deletion of fak (FAKK5 KO mice). FAK(K5 KO) mice displayed three major phenotypes--irregularities of hair cycle, sebaceous glands hypoplasia, and a thinner epidermis--pointing to defects in the proliferative capacity of multipotent stem cells found in the bulge. FAK-null keratinocytes in conventional primary culture undergo massive apoptosis hindering further analyses, whereas the defects observed in vivo do not shorten the mouse lifespan. These results suggest that the structure and the signaling environment of the native tissue may overcome the lack of signaling through FAK. Our findings point to the importance of in vivo and three-dimensional in vitro models in analyses of cell migration, proliferation, and survival. Surprisingly, the difference between FAKloxP/+ and FAKK5 KO mice in wound closure was not statistically significant, suggesting that in vivo loss of FAK does not affect migration/proliferation of basal keratinocytes in the same way as it affects multipotent stem cells of the skin.

Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development.

Background and aim We identified a balanced de novo translocation involving chromosomes Xq25 and 8q24 in an eight year-old girl with a non-progressive form of congenital ataxia, cognitive impairment and cerebellar hypoplasia. Methods and Results Breakpoint definition showed that the promoter of the Protein Tyrosine Kinase 2 ( PTK2 , also known as Focal Adhesion Kinase, FAK ) gene on chromosome 8q24.3 is translocated 2 kb upstream of the THO complex subunit 2 ( THOC2 ) gene on chromosome Xq25. PTK2 is a well-known non-receptor tyrosine kinase whereas THOC2 encodes a component of the evolutionarily conserved multiprotein THO complex, involved in mRNA export from nucleus. The translocation generated a sterile fusion transcript under the control of the PTK2 promoter, affecting expression of both PTK2 and THOC2 genes. PTK2 is involved in cell adhesion and, in neurons, plays a role in axonal guidance, and neurite growth and attraction. However, PTK2 haploinsufficiency alone is unlikely to be associated with human disease. Therefore, we studied the role of THOC2 in the CNS using three models: 1) THOC2 ortholog knockout in C.elegans which produced functional defects in specific sensory neurons; 2) Thoc2 knockdown in primary rat hippocampal neurons which increased neurite extension; 3) Thoc2 knockdown in neuronal stem cells (LC1) which increased their in vitro growth rate without modifying apoptosis levels. Conclusion We suggest that THOC2 can play specific roles in neuronal cells and, possibly in combination with PTK2 reduction, may affect normal neural network formation, leading to cognitive impairment and cerebellar congenital hypoplasia.

PURPOSE The lens is a powerful model system to study integrin-mediated cell-matrix interaction in an in vivo context, as it is surrounded by a true basement membrane, the lens capsule. To characterize better the function of integrin-linked kinase (ILK), we examined the phenotypic consequences of its deletion in the developing mouse lens. METHODS ILK was deleted from the embryonic lens either at the time of placode invagination using the Le-Cre line or after initial lens formation using the Nestin-Cre line. RESULTS Early deletion of ILK leads to defects in extracellular matrix deposition that result in lens capsule rupture at the lens vesicle stage (E13.5). If ILK was deleted at a later time-point after initial establishment of the lens capsule, rupture was prevented. Instead, ILK deletion resulted in secondary fiber migration defects and, most notably, in cell death of the anterior epithelium (E18.5-P0). Remarkably, dying cells did not stain positively for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or activated-caspase 3, suggesting that they were dying from a non-apoptotic mechanism. Moreover, cross to a Bax(fl/fl)/Bak⁻/⁻ mouse line that is resistant to most forms of apoptosis failed to promote cell survival in the ILK-deleted lens epithelium. Electron microscopy revealed the presence of numerous membranous vacuoles containing degrading cellular material. CONCLUSIONS. Our study reveals a role for ILK in extracellular matrix organization, fiber migration, and cell survival. Furthermore, to our knowledge we show for the first time that ILK disruption results in non-apoptotic cell death in vivo.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that has been extensively studied in fibroblasts; however its function in hematopoiesis remains an enigma. FAK is thought to be expressed in myeloid and erythroid progenitors, and its expression is enhanced in response to cytokines such as granu-locyte macrophage colony-stimulating factor. Furthermore, bone marrow cells cultured in granulocyte macrophage colony-stimulating factor show active migration and chemoattractant-induced polarization, which correlates with FAK induction. While loss of FAK in mice results in embryonic lethality, we have deleted FAK in the adult bone marrow. We show an essential role for FAK in regulating hemolytic, myelotoxic, as well as acute inflammatory stress responses in vivo. In vitro, loss of FAK in erythroid and myeloid progenitor's results in impaired cytokine induced growth and survival, as well as defects in the activation and expression of antiapoptotic proteins caspase 3 and Bcl-xL. Additionally, reduced migration and adhesion of myeloid cells on extracellular matrix proteins, as well as impaired activation of Rac GTPase is also observed in the absence of FAK. Our studies reveal an essential role for FAK in integrating growth/survival and adhesion based functions in myeloid and erythroid cells predominantly under conditions of stress.

The vertebrate lens provides an excellent model with which to study the mechanisms required for epithelial invagination. In the mouse, the lens forms from the head surface ectoderm. A domain of ectoderm first thickens to form the lens placode and then invaginates to form the lens pit. The epithelium of the lens placode remains in close apposition to the epithelium of the presumptive retina as these structures undergo a coordinated invagination. Here, we show that F-actin-rich basal filopodia that link adjacent presumptive lens and retinal epithelia function as physical tethers that coordinate invagination. The filopodia, most of which originate in the presumptive lens, form at E9.5 when presumptive lens and retinal epithelia first come into close contact, and have retracted by E11.5 when invagination is complete. At E10.5 - the lens pit stage - there is approximately one filopodium per epithelial cell. Formation of filopodia is dependent on the Rho family GTPase Cdc42 and the Cdc42 effector IRSp53 (Baiap2). Loss of filopodia results in reduced lens pit invagination. Pharmacological manipulation of the actin-myosin contraction pathway showed that the filopodia can respond rapidly in length to change inter-epithelial distance. These data suggest that the lens-retina inter-epithelial filopodia are a fine-tuning mechanism to assist in lens pit invagination by transmitting the forces between presumptive lens and retina. Although invagination of the archenteron in sea urchins and dorsal closure in Drosophila are known to be partly dependent on filopodia, this mechanism of morphogenesis has not previously been identified in vertebrates.

Focal adhesion kinase (FAK) is a central regulator of the focal adhesion, influencing cell proliferation, survival, and migration. Despite evidence demonstrating FAK overexpression in human cancer, its role in tumor initiation and progression is not well understood. Using Cre/LoxP technology to specifically knockout FAK in the mammary epithelium, we showed that FAK is not required for tumor initiation but is required for tumor progression. The mechanistic underpinnings of these results suggested that FAK regulates clinically relevant gene signatures and multiple signaling complexes associated with tumor progression and metastasis, such as Src, ERK, and p130Cas. Furthermore, a systems-level analysis identified FAK as a major regulator of the tumor transcriptome, influencing genes associated with adhesion and growth factor signaling pathways, and their cross talk. Additionally, FAK was shown to down-regulate the expression of clinically relevant proliferation- and metastasis-associated gene signatures, as well as an enriched group of genes associated with the G 2 and G 2 /M phases of the cell cycle. Computational analysis of transcription factor-binding sites within ontology-enriched or clustered gene sets suggested that the differentially expressed proliferation- and metastasis-associated genes in FAK-null cells were regulated through a common set of transcription factors, including p53. Therefore, FAK acts as a primary node in the activated signaling network in transformed motile cells and is a prime candidate for novel therapeutic interventions to treat aggressive human breast cancers.

Hematopoietic stem/progenitor cells (HSC/P) reside in the bone marrow in distinct anatomic locations (niches) to receive growth, survival and differentiation signals. HSC/P localization and migration between niches depend on cell-cell and cell-matrix interactions, which result from the cooperation of cytokines, chemokines and adhesion molecules. The CXCL12-CXCR4 pathway, in particular, is essential for myelopoiesis and B lymphopoiesis but the molecular mechanisms of CXCL12 action remain unclear. We previously noted a strong correlation between prolonged CXCL12-mediated focal adhesion kinase (FAK) phosphorylation and sustained pro-adhesive responses in progenitor B cells, but not in mature B cells. Although FAK has been well studied in adherent fibroblasts, its function in hematopoietic cells is not defined. We used two independent approaches to reduce FAK expression in (human and mouse) progenitor cells. RNA interference (RNAi)-mediated FAK silencing abolished CXCL12-induced responses in human pro-B leukemia, REH cells. FAK-deficient REH cells also demonstrated reduced CXCL12-induced activation of the GTPase Rap1, suggesting the importance of FAK in CXCL12-mediated integrin activation. Moreover, in FAK(flox/flox) hematopoietic precursor cells, Cre-mediated FAK deletion resulted in impaired CXCL12-induced chemotaxis. These studies suggest that FAK may function as a key intermediary in signaling pathways controlling hematopoietic cell lodgment and lineage development.

Focal adhesion kinase (FAK) is a ubiquitously expressed cytoplasmic tyrosine kinase strongly activated by integrins and neurohumoral factors. Previous studies have shown that cardiac FAK activity is enhanced by hypertrophic stimuli before the onset of overt hypertrophy. Herein, we report that conditional deletion of FAK from the myocardium of adult mice did not affect basal cardiac performance, myocyte viability, or myofibrillar architecture. However, deletion of FAK abolished the increase in left ventricular posterior wall thickness, myocyte cross-sectional area, and hypertrophy-associated atrial natriuretic factor induction following pressure overload. Myocyte-restricted deletion of FAK attenuated the initial wave of extracellular signal-regulated kinase activation and cFos expression induced by adrenergic agonists and biomechanical stress. In addition, we found that persistent challenge of mice with myocyte-restricted FAK inactivation leads to enhanced cardiac fibrosis and cardiac dysfunction in comparison to challenged genetic controls. These studies show that loss of FAK impairs normal compensatory hypertrophic remodeling without a concomitant increase in apoptosis in response to cardiac pressure overload and highlight the possibility that FAK activation may be a common requirement for the initiation of this compensatory response.

The process of cell migration is initiated by protrusion at the leading edge of the cell, the formation of peripheral adhesions, the exertion of force on these adhesions, and finally the release of the adhesions at the rear of the cell. Focal adhesion kinase (FAK) is intimately involved in the regulation of this process, although the precise mechanism(s) whereby FAK regulates cell migration is unclear. We have used two approaches to reduce FAK expression in fibroblasts. Treatment of cells with FAK-specific siRNAs substantially reduced FAK expression and inhibited the spreading of fibroblasts in serum-free conditions, but did not affect the rate of spreading in the presence of serum. In contrast with the wild-type cells, the FAK siRNA-treated cells exhibited multiple extensions during cell spreading. The extensions appeared to be inappropriately formed lamellipodia as evidenced by the localization of cortactin to lamellipodial structures and the inhibition of such structures by expression of dominant-negative Rac. The wild-type phenotype was restored by reexpressing wild-type FAK in the knockdown cells, but not by expression of FAK containing a point mutation at the autophosphorylation site (FAK Y397F). In wound-healing assays, FAK knockdown cells failed to form broad lamellipodia, instead forming multiple leading edges. Similar results were obtained using primary mouse embryo fibroblasts from FAK-flox mice in which Cre-mediated excision was used to ablate the expression of FAK. These data are consistent with a role for FAK in regulating the formation of a leading edge during cell migration by coordinating integrin signaling to direct the correct spatial activation of membrane protrusion.

Although netrins are an important family of neuronal guidance proteins, intracellular mechanisms that mediate netrin function are not well understood. Here we show that netrin-1 induces tyrosine phosphorylation of proteins including focal adhesion kinase (FAK) and the Src family kinase Fyn. Blockers of Src family kinases inhibited FAK phosphorylation and axon outgrowth and attraction by netrin. Dominant-negative FAK and Fyn mutants inhibited the attractive turning response to netrin. Axon outgrowth and attraction induced by netrin-1 were significantly reduced in neurons lacking the FAK gene. Our results show the biochemical and functional links between netrin, a prototypical neuronal guidance cue, and FAK, a central player in intracellular signaling that is crucial for cell migration.

The formation of neuronal networks in the central nervous system (CNS) requires precise control of axonal branch development and stabilization. Here we show that cell-specific ablation of the murine gene Ptk2 (more commonly known as fak), encoding focal adhesion kinase (FAK), increases the number of axonal terminals and synapses formed by neurons in vivo. Consistent with this, fak mutant neurons also form greater numbers of axonal branches in culture because they have increased branch formation and reduced branch retraction. Expression of wild-type FAK, but not that of several FAK variants that prevent interactions with regulators of Rho family GTPases including the p190 Rho guanine nuclear exchange factor (p190RhoGEF), rescues the axonal arborization phenotype observed in fak mutant neurons. In addition, expression of a mutant p190RhoGEF that cannot associate with FAK results in a phenotype very similar to that of neurons lacking FAK. Thus, FAK functions as a negative regulator of axonal branching and synapse formation, and it seems to exert its actions, in part, through Rho family GTPases., This work was supported by a grant from the NIH and by the Howard Hughes Medical Institute (HHMI). B.R. was supported by a grant from the Ministerio de Educación y Cultura of Spain and by the HHMI. L.F.R. is an Investigator of the HHMI. B.R. is a Ramón y Cajal Investigator from Ministerio de Ciencia y Tecnología of Spain.

Axonal growth cones respond to adhesion molecules and extracellular matrix components by rapid morphological changes and growth rate modification. Neurite outgrowth mediated by the neural cell adhesion molecule (NCAM) requires the src family tyrosine kinase p59(fyn) in nerve growth cones, but the molecular basis for this interaction has not been defined. The NCAM140 isoform, which is found in migrating growth cones, selectively co-immunoprecipitated with p59(fyn) from nonionic detergent (Brij 96) extracts of early postnatal mouse cerebellum and transfected rat B35 neuroblastoma and COS-7 cells. p59(fyn) did not associate significantly with the NCAM180 isoform, which is found at sites of stable neural cell contacts, or with the glycophosphatidylinositol-linked NCAM120 isoform. pp60(c-)src, a tyrosine kinase that promotes neurite growth on the neuronal cell adhesion molecule L1, did not interact with any NCAM isoform. Whereas p59(fyn) was constitutively associated with NCAM140, the focal adhesion kinase p125(fak), a nonreceptor tyrosine kinase known to mediate integrin-dependent signaling, became recruited to the NCAM140-p59(fyn) complex when cells were reacted with antibodies against the extracellular region of NCAM. Treatment of cells with a soluble NCAM fusion protein or with NCAM antibodies caused a rapid and transient increase in tyrosine phosphorylation of p125(fak) and p59(fyn). These results suggest that NCAM140 binding interactions at the cell surface induce the assembly of a molecular complex of NCAM140, p125(fak), and p59(fyn) and activate the catalytic function of these tyrosine kinases, initiating a signaling cascade that may modulate growth cone migration.

Abstract 1119 Progenitor B cells are thought to reside in complex bone marrow microenvironments, e.g. niches, where they receive signals for growth and maturation. Here we have conditionally targeted in B cells the non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK), because of its function as an integrator of cell extrinsic signals including CXCL12 and VCAM-1. The number of progenitor (pro-, pre- and immature) B cells is reduced by 30–40% in CD19-Cre Fak fl/fl mice compared to wild type mice, and ex vivo cultured, Fak deleted pro-B cells exhibit impaired IL-7 ± CXCL12 mediated proliferation and survival. A novel quantitative laser scanning cytometry approach was used to demonstrate that Fak deletion also disrupts the non-random gradient distribution in femoral bone marrow of progenitor B cells, which normally are preferentially localized in endosteal regions of the metaphyses and to a lesser degree of the diaphysis. Increased numbers of pro-B cells are detected in the periphery of Fak deleted mice, and mobilization of pro-B cells induced by the inflammatory antigen NP-CGG-alum is increased 3-fold in Fak-deleted versus wild type mice. In addition, intravenously transferred Fak deleted pro-B cells have defective homing specifically to the extra-vascular compartment of the bone marrow. Collectively, these studies illustrate the importance of FAK in regulating progenitor B cell homeostasis and maintenance of their spatial distribution in bone marrow niches. Disclosures: No relevant conflicts of interest to declare.

Introduction: Synovial fibroblasts invade cartilage and bone, leading to joint destruction in rheumatoid arthritis. However, the mechanisms that regulate synovial fibroblast invasion are not well understood. Focal adhesion kinase (FAK) has been implicated in cellular invasion in several cell types, and FAK inhibitors are in clinical trials for cancer treatment. Little is known about the role of FAK in inflammatory arthritis, but, given its expression in synovial tissue, its known role in invasion in other cells and the potential clinical availability of FAK inhibitors, it is important to determine if FAK contributes to synovial fibroblast invasion and inflammatory arthritis. Methods: After treatment with FAK inhibitors, invasiveness of human rheumatoid synovial fibroblasts was determined with Matrigel invasion chambers. Migration and focal matrix degradation, two components of cellular invasion, were assessed in FAK-inhibited rheumatoid synovial fibroblasts by transwell assay and microscopic examination of fluorescent gelatin degradation, respectively. Using mice with tumor necrosis factor α (TNFα)–induced arthritis in which fak could be inducibly deleted, invasion and migration by FAK-deficient murine arthritic synovial fibroblasts were determined as described above and arthritis was clinically and pathologically scored in FAK-deficient mice. Results: Inhibition of FAK in human rheumatoid synovial fibroblasts impaired cellular invasion and migration. Focal matrix degradation occurred both centrally and at focal adhesions, the latter being a novel site for matrix degradation in synovial fibroblasts, but degradation was unaltered with FAK inhibitors. Loss of FAK reduced invasion in murine arthritic synovial fibroblasts, but not migration or TNFα-induced arthritis severity and joint erosions. Conclusions: FAK inhibitors reduce synovial fibroblast invasion and migration, but synovial fibroblast migration and TNFα-induced arthritis do not rely on FAK itself. Thus, inhib ition of FAK alone is unlikely to be sufficient to treat inflammatory arthritis, but current drugs that inhibit FAK may inhibit multiple factors, which could increase their efficacy in rheumatoid arthritis.