Focal adhesion kinase inhibition decreases cell viability and induces apoptosis of JAK2 V617F positive cells

Abstract Focal Adhesion Kinase (FAK) protein participates in proliferation, migration, cell survival, and apoptosis process. It has been described as overexpressed in several neoplasms being a promising target for therapy. BCR-ABL negative chronic Myeloproliferative Neoplasms (MPN) are clonal disorders characterized by the excess of proliferation and apoptosis resistance. The identification of the acquired JAK2 V617F mutation in MPN patients allowed a better understanding of pathogenesis. However, there is still no pharmacological treatment that leads all patients to molecular remission, justifying new studies. The present study aimed to evaluate FAK involvement in the viability and apoptosis of HEL and SET-2 cells, both JAK2 V617F positive cell lines. The FAK inhibitor PF 562,271 was used. Cell viability was determined using MTT assay and apoptosis verified by cleaved PARP, cleaved Caspase 3 and Annexin-V/PI staining detection. FAK inhibition significantly reduced HEL and SET-2 cells viability and induced apoptosis. Considering the role of JAK/STAT pathway in MPN, further investigation of FAK participation in the MPN cells proliferation and apoptosis resistance, as well as possible crosstalk between JAK and FAK and downstream pathways may contribute to the knowledge of MPN pathophysiology, the discovery of new molecular targets, and JAK inhibitors resistance mechanisms.

Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies.

OBJECTIVE: The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS). METHODOLOGY: Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively. RESULTS: FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces. CONCLUSIONS: Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies

Abstract Objective The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS). Methodology Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively. Results FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces. Conclusions Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

Substrate Stiffness-Dependent Carbon Nanotube-Induced Lung Fibrogenesis.

Most living tissues exhibit the specific stiffness, which has been known to have profound influence on cell behaviors, yet how the stiffness affects cellular responses to engineered nanomaterials has not been elucidated. Particularly, discrepancies exist between in vitro and in vivo nanotoxicological studies. Here, we investigated the effects of substrate stiffness on the fibrogenic responses of normal human lung fibroblasts (NHLFs) to multiwalled carbon nanotubes (MWCNTs). NHLFs were grown on polyacrylamide (PAAm) hydrogels with the stiffness comparable to that of human normal and fibrotic lung tissues, and treated with MWCNTs for various time. The fibrogenic responses, including cell proliferation, reactive oxygen species production, and collagen I expression, of NHLFs to MWCNTs were observed to be regulated by substrate stiffness in a time-dependent manner. NHLFs generally were rounded on soft hydrogels and required a long treatment time to exhibit fibrogenic responses, while on stiff hydrogels the cells were well-spread with defined stress fibers and short-time MWCNTs treatment sufficiently induced the fibrogenic responses. Mechanistic studies showed that MWCNTs induced fibrogenesis of NHLFs through promoting expression and phosphorylation of focal adhesion kinase (FAK), while attenuating intracellular tension in the cells on stiff gels could increase MWCNTs uptake and thus elevate the induced fibrogenic responses. Moreover, we proposed a time-stiffness superposition principle to describe the equivalent effects of treatment time and substrate stiffness on nanomaterials-induced fibrogenesis, which suggested that increasing substrate stiffness expedited fibrogenesis and shed light on the rational design of in vitro models for nanotoxicological study.

Leishmania infection inhibits macrophage motility by altering F-actin dynamics and the expression of adhesion complex proteins.

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis-infected macrophages also show reduced directional migration in response to the chemokine MCP-1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F-actin turnover frequency in L. amazonensis-infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane-extracellular matrix interactions.

Activation of focal adhesion kinase via M1 muscarinic acetylcholine receptor is required in restitution of intestinal barrier function after epithelial injury.

Impairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remain to be clarified. In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation were also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions.

The role of focal adhesion kinase in the regulation of cellular mechanical properties.

The regulation of mechanical properties is necessary for cell invasion into connective tissue or intra- and extravasation through the endothelium of blood or lymph vessels. Cell invasion is important for the regulation of many healthy processes such as immune response reactions and wound healing. In addition, cell invasion plays a role in disease-related processes such as tumor metastasis and autoimmune responses. Until now the role of focal adhesion kinase (FAK) in regulating mechanical properties of cells and its impact on cell invasion efficiency is still not well known. Thus, this review focuses on mechanical properties regulated by FAK in comparison to the mechano-regulating protein vinculin. Moreover, it points out the connection between cancer cell invasion and metastasis and FAK by showing that FAK regulates cellular mechanical properties required for cellular motility. Furthermore, it sheds light on the indirect interaction of FAK with vinculin by binding to paxillin, which then impairs the binding of paxillin to vinculin. In addition, this review emphasizes whether FAK fulfills regulatory functions similar to vinculin. In particular, it discusses the differences and the similarities between FAK and vinculin in regulating the biomechanical properties of cells. Finally, this paper highlights that both focal adhesion proteins, vinculin and FAK, synergize their functions to regulate the mechanical properties of cells such as stiffness and contractile forces. Subsequently, these mechanical properties determine cellular invasiveness into tissues and provide a source sink for future drug developments to inhibit excessive cell invasion and hence, metastases formation.

PTK2 and PTPN11 expression in myelodysplastic syndromes.

OBJECTIVE: The aim of this study was to evaluate the expression of protein tyrosine kinase 2 and protein tyrosine phosphatase non-receptor type 11, which respectively encode focal adhesion kinase protein and src homology 2 domain-containing protein-tyrosine phosphatase 2, in hematopoietic cells from patients with myelodysplastic syndromes. METHODS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions were analyzed by quantitative polymerase chain reaction in bone marrow cells from patients with myelodysplastic syndromes and healthy donors. RESULTS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions did not significantly differ between normal cells and myelodysplastic cells. CONCLUSIONS: Our data suggest that despite the relevance of focal adhesion kinase and src homology 2 domain-containing protein-tyrosine phosphatase 2 in hematopoietic disorders, their mRNA expression do not significantly differ between total bone marrow cells from patients with myelodysplastic syndromes and healthy donors.

PTK2 and PTPN11 expression in myelodysplastic syndromes

OBJECTIVE: The aim of this study was to evaluate the expression of protein tyrosine kinase 2 and protein tyrosine phosphatase non-receptor type 11, which respectively encode focal adhesion kinase protein and src homology 2 domain-containing protein-tyrosine phosphatase 2, in hematopoietic cells from patients with myelodysplastic syndromes. METHODS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions were analyzed by quantitative polymerase chain reaction in bone marrow cells from patients with myelodysplastic syndromes and healthy donors. RESULTS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions did not significantly differ between normal cells and myelodysplastic cells. CONCLUSIONS: Our data suggest that despite the relevance of focal adhesion kinase and src homology 2 domain-containing protein-tyrosine phosphatase 2 in hematopoietic disorders, their mRNA expression do not significantly differ between total bone marrow cells from patients with myelodysplastic syndromes and healthy donors. .

[Effect of tyrosine kinase inhibitor imatinib mesylate on K562 cell invasion by PTEN pathway].

AIM: To investigate the effect of tyrosine kinase inhibitor imatinib mesylate on the PTEN signaling pathway and the cell invasion in K562 cells. METHODS: K562 cells were treated with different concentrations of imatinib mesylate. After different time periods, the mRNA levels of BCR/ABL, PTEN and FAK were detected by real-time fluorescent quantitative PCR (FQ-PCR) to analyze their relationships. The protein level of FAK was detected by immunocytochemistry. The cell invasive ability was examined by Transwell (Boyden chamber) assay. RESULTS: In the initial 36 h, the expression level of PTEN mRNA was up-regulated and the FAK mRNA was down-regulated with the reduction of BCR/ABL fusion gene expression and the cell invasive ability of K562 cells was inhibited by 2 µg/mL imatinib mesylate. 48 h later, the PTEN mRNA expression level decreased and the FAK mRNA expression level was elevated with the restore of BCR/ABL fusion gene. BCR/ABL mRNA level presented a positive correlation with PTEN mRNA expression level, and a negative correlation with FAK mRNA. CONCLUSION: Tyrosine kinase inhibitor imatinib mesylate can regulate PTEN/FAK pathway and inhibit the leukemia K562 cell invasive ability via restraining BCR/ABL fusion gene.

Focal adhesion kinase signaling is necessary for the Cyclosporin A-enhanced migration and invasion of human trophoblast cells.

OBJECTIVES: Our previous studies have shown that Cyclosporin A (CsA) promotes human trophoblast invasion via mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. E-cadherin and matrix metalloproteinases (MMPs) are important mediators in trophoblast migration and invasion. Here, we further investigate the role of focal adhesion kinase (FAK) signaling in the CsA-induced trophoblast migration and invasion and ERK activation. STUDY DESIGN: The migration and invasion of human primary trophoblasts and JEG-3 cells were measured by transwell migration and matrigel invasion assays. The activation of FAK, Src and ERK induced by CsA were examined by western blot. The colocalization of FAK and Src was detected by dual immunofluorescence assay. The regulation of E-cadherin expression and matrix metalloproteinases (MMPs) activity was evaluated by western blot and gelatin zymography, respectively. RESULTS: CsA increased the phosphorylation of FAK and Src in human primary trophoblasts and JEG-3 cells. Meanwhile, the activated FAK and Src colocalized in the cytoplasm of JEG-3 cells. The FAK inhibitor Y15 or Src inhibitor PP2 could abrogate the phosphorylation of ERK, the enhanced migration and invasion and the activity of MMP2, 9 induced by CsA. In addition, these inhibitors also restored the expression of E-cadherin which is down-regulated by CsA. However, U0126, an inhibitor of ERK, had no significant effect on the CsA-induced activation of FAK and Src. CONCLUSIONS: FAK-Src signaling, the upstream signaling cascade of ERK activation, plays an important role in the CsA-induced migration and invasion via down-regulating expression of E-cadherin and up-regulating activity of MMP2, 9 in trophoblast cells. These results may help provide a rationale to develop a novel therapeutic strategy for pregnancy disorders from insufficient trophoblast invasion.

Time-resolved fluorescence resonance energy transfer as a versatile tool in the development of homogeneous cellular kinase assays.

Homogeneous cellular assays can streamline product detection in the drug discovery process. One commercially available assay employing time-resolved fluorescence resonance energy transfer (TR-FRET) that detects phosphorylated products was used to evaluate inhibitors of the receptor tyrosine kinase AXL in a cell line expressing an AXL-green fluorescent protein fusion protein. This TR-FRET assay was modified to evaluate the phosphorylation state of the AXL family member MER in a cell line expressing MER with a V5 tag by adding a fluorescein-labeled anti-V5 antibody. This homogeneous cellular assay was further modified to evaluate the nonreceptor tyrosine kinase focal adhesion kinase (FAK) in cell lines that expressed an untagged kinase by the inclusion of a commercially available anti-FAK antibody conjugated with an acceptor dye. The methods described here can be further adapted for TR-FRET detection of other cellular kinase activities.

Retinal pigment epithelial cells use a MerTK-dependent mechanism to limit the phagocytic particle binding activity of αvß5 integrin.

BACKGROUND INFORMATION: αvß5 integrin and Mer tyrosine kinase (MerTK) receptors reside at the apical surface of the retinal pigment epithelium (RPE) in the eye to promote the diurnal, synchronised phagocytosis of shed photoreceptor outer segment fragments (POS) that is critical for vision. Phagocytosis assays studying RPE cells in culture have defined roles for αvß5 in POS surface binding and for MerTK in engulfment of bound POS. Both receptors have thus far only been studied separately. It is therefore unknown if αvß5 integrin activity in POS binding is independent of the engulfment function of RPE cells. This study investigates how increasing αvß5 receptor levels affect POS binding and internalisation by wild-type (wt), αvß5- or MerTK-deficient RPE. RESULTS: ß5 integrin-green fluorescent protein (ß5-GFP) fusion proteins formed heterodimeric receptors with endogenous αv integrin subunits at the apical surface of mouse or rat RPE cells that co-immunoprecipitated focal adhesion kinase and redistributed with bound POS such as endogenous αvß5 receptors. In ß5(-/-) RPE cells, de novo formation of αvß5-GFP receptors restored POS binding and internalisation up to, but not, above wt POS uptake levels. In wt RPE cells, increasing levels of αvß5 surface receptors by over-expressing ß5-GFP only moderately stimulated POS binding, even if POS internalisation was inhibited pharmacologically or by lowering incubation temperatures. In contrast, the same increase in αvß5 receptor levels dramatically enhanced POS binding of RPE cells lacking MerTK. Furthermore, decreasing MerTK expression by RNA interference increased POS binding to endogenous αvß5 receptors of wt RPE cells. CONCLUSIONS: Expressing ß5-GFP is sufficient to reverse phagocytic deficiencies of RPE cells derived from ß5(-/-) mice, indicating that these cells do not irreversibly lose other components of the phagocytic machinery. RPE cells expressing the engulfment receptor MerTK control POS binding by limiting activity of endogenous αvß5 and αvß5-GFP integrins, although they reside at the apical, phagocytic surface. In contrast, RPE cells permanently or transiently losing MerTK expression lack this regulatory mechanism and bind excess POS via surface αvß5 receptors. Taken together, these data reveal a novel feedback mechanism that restricts binding of POS to surface αvß5 integrin receptors in RPE cells.

Biological behavior of fibroblast-like cells cultured on anodized-hydrothermally treated titanium with a nanotopographic surface structure.

PURPOSE: The interface between the transmucosal portion of endosseous implants surface and the connective tissue is characterized by fibroblast-rich barrier tissue, which is important for the long-term stability and maintenance of the implant. This study investigated the effect of cell adhesion on focal adhesion kinase (FAK) protein and on gene expression over a 72-h culture period. Fibroblast-like cells were cultured on anodized-hydrothermally treated commercially pure titanium with nanotopographic structure (SA-treated c.p.Ti) surfaces. METHODS: Murine fibroblast-like NIH/3T3 cells were cultured for 10-72h on c.p.Ti, anodic oxide (AO) c.p.Ti, and SA-treated c.p.Ti disks. Cell morphology was analyzed using scanning electron microscopy (SEM). Cytoskeletal structure and FAK protein localization were analyzed using confocal laser scanning microscopy (CLSM). FAK mRNA levels were analyzed using real-time quantitative RT-PCR. RESULTS: SEM and CLSM showed increased NIH/3T3 cell adhesion with time, and actin filaments oriented parallel with the filopodium-like extensions on all disks. Filopodium-like extensions were bound tightly to the nanotopographic structure surface of cultures on SA-treated c.p.Ti, and especially at 72h. FAK protein was localized along cellular extensions on SA-treated c.p.Ti and the expression of FAK mRNA was significantly higher on these disks than on c.p.Ti and AO c.p.Ti after 72h (P<0.05). CONCLUSIONS: NIH/3T3 fibroblast-like cells have the capacity to adhere to SA-treated c.p.Ti as a transmucosal portion of implant surface material and express focal adhesion molecules, which may play a key role in the maintenance of a mucosal tissue barrier.

Rho-Rho kinase pathway in the actomyosin contraction and cell-matrix adhesion in immortalized human trabecular meshwork cells.

PURPOSE: The outflow facility for aqueous humor across the trabecular meshwork (TM) is enhanced by agents that oppose the actomyosin contraction of its resident cells. Phosphorylation of MYPT1 (myosin light chain [MLC] phosphatase complex of Type 1) at Thr853 and Thr696 inhibits dephosphorylation of MLC, leading to an increase in actomyosin contraction. In this study, we examined the effects of Rho kinase (ROCK) inhibitors on the relative dephosphorylation of the two sites of MYPT1 using human TM cells (GTM3). METHODS: Dephosphorylation of MYPT1 at Thr853 and Thr696 was determined by western blot analysis following exposure to selective inhibitors of ROCK, namely Y-27632 and Y-39983. Consequent dephosphorylation of MLC and decreases in actomyosin contraction were assessed by western blot analysis and collagen gel contraction assay, respectively. Changes in the cell-matrix adhesion were measured in real time by electric cell-substrate impedance sensing and also assessed by staining for paxillin, vinculin, and focal adhesion kinase (FAK). RESULTS: Both ROCK inhibitors produced a concentration-dependent dephosphorylation at Thr853 and Thr696 of MYPT1 in adherent GTM3 cells. IC50 values for Y-39983 were 15 nM and 177 nM for dephosphorylation at Thr853 and Thr696, respectively. Corresponding values for Y-27632 were 658 nM and 2270 nM. Analysis of the same samples showed a decrease in MLC phosphorylation with IC50 values of 14 nM and 1065 nM for Y-39983 and Y-27632, respectively. Consistent with these changes, both inhibitors opposed contraction of collagen gels induced by TM cells. Exposure of cells to the inhibitors led to a decrease in the electrical cell-substrate resistance, with the effect of Y-39983 being more pronounced than Y-27632. Treatment with these ROCK inhibitors also showed a loss of stress fibers and a concomitant decrease in tyrosine phosphorylation of paxillin and FAK. CONCLUSIONS: Y-39983 and Y-27632 oppose ROCK-dependent phosphorylation of MYPT1 predominantly at Thr853 with a corresponding decrease in MLC phosphorylation. A relatively low effect of both ROCK inhibitors at Thr696 suggests a role for other Ser/Thr kinases at this site. Y-39983 was several-fold more potent when compared with Y-27632 at inhibiting the phosphorylation of MYPT1 at either Thr853 or Thr696 commensurate with its greater potency at inhibiting the activity of human ROCK-I and ROCK-II enzymes.

Expression of focal adhesion proteins in the developing rat kidney.

Focal adhesions play a critical role as centers that transduce signals by cell-matrix interactions and regulate fundamental processes such as proliferation, migration, and differentiation. Focal adhesion kinase (FAK), paxillin, integrin-linked kinase (ILK), and hydrogen peroxide-inducible clone-5 (Hic-5) are major proteins that contribute to these events. In this study, we investigated the expression of focal adhesion proteins in the developing rat kidney. Western blotting analysis revealed that the protein levels of FAK, p-FAK(397), paxillin, p-paxillin(118), and Hic-5 were high in embryonic kidneys, while ILK expression persisted from the embryonic to the mature stage. Immunohistochemistry revealed that FAK, p-FAK(397), paxillin, and p-paxillin(118) were strongly expressed in condensed mesenchymal cells and the ureteric bud. They were detected in elongating tubules and immature glomerular cells in the nephrogenic zone. Hic-5 was predominantly expressed in mesenchymal cells as well as immature glomerular endothelial and mesangial cells, suggesting that Hic-5 might be involved in mesenchymal cell development. ILK expression was similar to that of FAK in the developmental stages. Interestingly, ILK was strongly expressed in podocytes in mature glomeruli. ILK might play a role in epithelial cell differentiation as well as kidney growth and morphogenesis. In conclusion, the temporospatially regulated expression of focal adhesion proteins during kidney development might play a role in morphogenesis and cell differentiation.

MPC polymer regulates fibrous tissue formation by modulating cell adhesion to the biomaterial surface.

The aim of this study was to analyze the effects of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer on fibrous tissue formation and cell adhesion plaque (CAP)-forming reactions. Silastic elastomer (SE) plates coated (experimental group) and uncoated (control group) with MPC polymer were prepared for in vivo and in vitro experiments. For the in vivo animal experiments, SE plates were implanted subcutaneously in the rat dorsal region. At 4, 8, and 12 weeks, thicknesses of the fibrous tissue capsules in the experimental group were lower than in the control group. Likewise, the amount of collagen in the experimental group was lower than that of the control group. For the in vitro cell culture experiments, KMST-6 fibroblast cells in the experimental group demonstrated enhanced cell migration, accompanied with a weaker expression of vinculin and a larger amount of filopodia. Furthermore, weaker expressions of paxillin, talin, and ROCK1, but stronger expression of cofilin, were observed in the experimental group. Taken together, these results suggested that MPC polymer regulated fibrous tissue formation by modulating cell adhesion through changes in local CAPs and downstream signaling.

DNA copy number aberrations in small-cell lung cancer reveal activation of the focal adhesion pathway.

Small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancer in its clinical behavior, with a 5-year overall survival as low as 5%. Despite years of research in the field, molecular determinants of SCLC behavior are still poorly understood, and this deficiency has translated into an absence of specific diagnostics and targeted therapeutics. We hypothesized that tumor DNA copy number alterations would allow the identification of molecular pathways involved in SCLC progression. Array comparative genomic hybridization was performed on DNA extracted from 46 formalin-fixed paraffin-embedded SCLC tissue specimens. Genomic profiling of tumor and sex-matched control DNA allowed the identification of 70 regions of copy number gain and 55 regions of copy number loss. Using molecular pathway analysis, we found a strong enrichment in these regions of copy number alterations for 11 genes associated with the focal adhesion pathway. We verified these findings at the genomic, gene expression and protein level. Focal Adhesion Kinase (FAK), one of the central genes represented in this pathway, was commonly expressed in SCLC tumors and constitutively phosphorylated in SCLC cell lines. Those were poorly adherent to most substrates but not to laminin-322. Inhibition of FAK phosphorylation at Tyr(397) by a small-molecule inhibitor, PF-573,228, induced a dose-dependent decrease of adhesion and an increase of spreading in SCLC cell lines on laminin-322. Cells that tended to spread also showed a decrease in focal adhesions, as demonstrated by a decreased vinculin expression. These results support the concept that pathway analysis of genes in regions of copy number alterations may uncover molecular mechanisms of disease progression and demonstrate a new role of FAK and associated adhesion pathways in SCLC. Further investigations of FAK at the functional level may lead to a better understanding of SCLC progression and may have therapeutic implications.

Effects of epidermal growth factor and/or nerve growth factor on Malassez's epithelial rest cells in vitro: expression of mRNA for osteopontin, bone morphogenetic protein 2 and vascular endothelial growth factor.

BACKGROUND AND OBJECTIVE: Malassez's epithelial rest (MER) cells are involved in the maintenance and homeostasis of the periodontal ligament (PDL). The purpose of this study was to determine the effects of epidermal growth factor (EGF) and/or nerve growth factor (NGF) in vitro on these functions of MER cells. MATERIAL AND METHODS: MER cells from porcine PDL were incubated for 3 or 9 h after the addition of EGF and/or NGF to final concentrations of 10 ng/mL. Cells cultured without those growth factors were used as controls. The expression of mRNA for osteopontin, bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF) was analyzed using quantitative RT-PCR. RESULTS: There was a decrease in the expression of osteopontin mRNA by MER cells treated for 9 h with NGF and the level of mRNA expressed was lower than that of the control and EGF-treated groups. The expression of BMP-2 mRNA by MER cells treated with NGF for 9 h also decreased, and was lower than that of the control and EGF-treated groups. The expression of VEGF mRNA by MER cells treated with EGF for 3 or 9 h was higher than in the control and NGF-treated groups. The expression of VEGF mRNA was lower in MER cells treated with NGF for 3 and 9 h than in the control and EGF-treated groups, and decreased from 3 to 9 h of treatment. EGF stimulated MER cells to secrete VEGF, which suggests that EGF plays an important role in maintaining the homeostasis of the PDL. NGF acts on MER cells to inhibit calcification in the PDL. Furthermore, in the EGF+NGF-treated MER cells, expression of mRNA for BMP-2 and VEGF was similar to that of the NGF-treated group, but cell proliferation and expression of osteopontin mRNA were similar to that of the EGF-treated group. CONCLUSION: EGF and NGF play important roles in maintaining the PDL.

Immunohistochemical study of heat shock protein 27 with respect to survival and regeneration of proximal tubular cells after uranyl acetate-induced acute tubular injury in rats.

This study examined the possible role of heat shock protein 27 (HSP27) expression in the survival and regeneration of proximal tubule (PT) cells after acute tubular injury. Rats were injected with a low (0.2 mg/kg) or high (4 mg/kg) dose of uranyl acetate (UA) to induce renal injury. Renal tissues were immunostained for HSP27, focal adhesion kinase (FAK), and bromodeoxyuridine (BrdU), and stained by the TUNEL method. Low-dose UA induced focal PT depletion in the proximal three-quarters of the S3 segment. Here, cells became sporadically positive for cytoplasmic HSP27 in association with FAK+, and almost all BrdU+ early regenerating cells were positive for HSP27 from days 2 to 3. High-dose UA induced severe PT depletion in the proximal three-quarters of S3, and a small number of PT cells became positive for HSP27 as early as day 2. BrdU+, early regenerating cells were restricted to the distal quarter of S3 from days 2 to 3, with or without HSP27 staining and with FAK. In both groups, HSP+ PT cells and BrdU+ cells peaked in number at day 5. The PT cells showed reduced HSP27 accumulation by day 7 as they differentiated, but remained immunopositive for FAK. TUNEL+ apoptotic cells were immunonegative for both HSP27 and FAK. Cytoplasmic HSP27 accumulation in PT cells seems to contribute to PT survival and transition from PT cell proliferation to differentiation. When PT cells are severely impaired, distinct cells in the distal areas of S3 could undergo cell cycle progression without HSP27 accumulation.