Establishment of FAK null osteoblast and bone marrow cell lines. (A) In vitro knockout of fak in osteoblasts. Cells from floxed animals (FAK^fl/fl;p53^−/−) were treated with adenovirus carrying cre. One microgram of total protein lysates was analyzed by Western blot using a monoclonal antibody against Cre. Signal was detected using enhanced chemiluminescence. After 5 days, the Cre protein level was tapered off. The same lysates were subjected to Western blot using a monoclonal antibody against FAK. FAK level was significantly reduced after 5 days of infection. (B) Establishment of FAK null osteoblast clonal lines. One microgram of total protein from immortalized calvarial osteoblasts was analyzed using monoclonal antibody against FAK. β-actin was used to ensure an equal amount of protein loading. +; positive control (FAK^fl/fl;p53^−/−), lane 1-6; FAK null osteoblast clonal lysates. (C,D) Calvarial osteoblast (C) and bone marrow cell line (D) differentiation in vitro. Immortalized calvarial FAK null clones were induced to differentiate in vitro. Cells were grown to confluency and differentiation was induced using ascorbic acid and β-glycerophosphate. After 2 weeks, cells were stained with 0.2% Alizarin Red to detect mineralization. A portion of FAK null clones (30%) differentiated in vitro. Mineralized and non-mineralized clones were labeled in red and green respectively. FAK^fl/fl; parental cell line (FAK^fl/fl;p53^−/−), −AA; uninduced, +AA: induced by ascorbic acid and β -glycerophosphate; OB: osteoblast; BM; bone marrow.

In situ hybridization for FAK in bone tissues. (A) Diagram of FAK protein and cDNA probe regions. FRNK is initiated at a methionine 668 and results in a truncated FAK protein. (B-E) In situ hybridization for N-terminal (B,C) and C-terminal of FAK transcripts (D,E) in wildtype and FAK mutant tibiae. FAK mutants were void of the transcripts for the N-terminal of FAK (C), while they expressed transcripts for the C-terminal of FAK transcripts (E). Bar: 100 μm.

Delayed bone healing in FAK mutants. Mono-cortical tibial injuries were induced in wild type and FAK mutants. (A-H) Histology of wild type and FAK mutant injury at post-surgical d7, 14, 21 and 28. FAK mutants showed delayed skeletal healing compared to wild type animals. At 28 days after surgery, FAK mutants developed exuberant calluses compared to wild type counterparts. (I,J) Alkaline phosphatase activity in the injury sites at post-surgical d7. FAK mutants (J) showed less enzyme activity in the injury site compared to wild type animals (I). (K,L) PECAM staining at post-surgical d7 showed equivalent number of endothelial cells in both wild type (K) and FAK mutant (L). (M,N) PCNA staining revealed a similar proliferative activity in wild type and FAK mutant. c: cortex, is: injury site. Bar: 100 μm.

Western and Immunofluorescence analyses of FAK and Pyk2 in FAK null osteoblast cell lines. (A) Western blot of total Pyk2 protein in FAK null osteoblast clones (FAK^−/−;p53^−/−). A total of 2 μg of cell lysate was loaded in each lane. Membrane was blotted with Pyk2 antibody (A) and phosphospecific Pyk2 antibody (B). β-actin was used for a loading control. The activated form of Pyk2 was increased in FAK null cells. (C,E) Immunofluorescence of total Pyk2 (C) and FAK (E) in wild type (FAK^fl/fl;p53^−/−) osteoblast cells. Pyk2 was found in the perinuclear region in wild type cells. (D,F) Immunofluorescence analyses of total Pyk2 (D) and phosphospecific Pyk2 (F) in FAK null osteoblast cells (FAK^−/−;p53^−/−). Inset in panel D; high magnification of focal adhesions. When FAK was deleted, Pyk2 was redistributed into focal adhesions in FAK null cells. Active form of Pyk2 was found in focal adhesions in FAK null cells. Bar; 10 μm.

Cre-mediated recombination of fak and skeletal pattern in FAK mutants. (A) Genomic structure of floxed fak mouse. When the floxed gene is recombined by Cre recombinase, the distance between the P1 and P2 primer is shortened. Floxed and recombined alleles generate 1.6kbp and 327bp PCR products respectively. (B) PCR analysis of Cre-mediated recombination in the animals. Typical screening gel from tail clips. fl; floxed allele, rec; recombined allele by Cre recombinase. (C,D) Cross section of humeri at e14.5 of wild type mouse (C) and FAK mutant (D) were collected and processed for Movat's pentachrome staining. Both wild type and FAK mutant showed the same pattern of chondrogenesis in limb development. (E,F) Whole mount bone and cartilage staining of wild type and FAK mutant. Wild type P5 mouse (E) and FAK mutant (F) were stained with Alizarin Red and Alcian Blue. Skeletal system in FAK mutant was not affected. (G,H) Longitudinal sections of mouse tibiae from wild type (G) and FAK mutant (H). Tibiae from three month old wild type and FAK mutant showed well developed growth plates. fi: fibula, ti: tibia, gp: growth plate, oc: ossification center. Bar: 100 μm.

Molecular analyses of osteogenic genes in wild type animals and Cre expression in the Col1-Cre mouse. (A) In situ hybridization of wild type tibia for collagen type I. Col I was expressed in osteocytes (arrows) and in the cambial layer of the periosteum. (B) Immunohistochemistry for Cre in the tibia of the Col1-Cre mouse. Cre protein was detected in the periosteum. (C,D) In situ hybridization for runx2 (C) and sox9 (D) in wild type tibia. Sox9 was expressed in both periosteum and osteocytes (arrows). (E,F) Collagen type I (col I) in situ hybridization and Cre immunohistochemistry in the pinhole injury sites from FAK mutants at d7. Col I and Cre expression was colocalized in the injury site. (E). c: cortex, is: injury site, po: periosteum. Bar: 100 μm.

Defective osteoclast activity in FAK mutant mice. (A-D) TRAP staining of the injury sites in wild type (A,C) and FAK mutants (B,D) at post-surgical d7 and 28. Wild type animals showed higher TRAP activity throughout the healing process than FAK mutants. (E-F) Remodeling of bone chips [75] in wild type and FAK mutants at 7 days post-surgery. (E) Wild type bone chips in a wild type injury site. (F) Wild type bone chips in a FAK mutant. (G) FAK mutant bone chips in a wild type animal. (H) FAK mutant bone chips into a FAK mutant. Osteoclast activity in the animals that received FAK mutant bone chips did not activate bone remodeling. (I, J) Double staining of TRAP activity and nuclei in the bone grafted injury sites. (I) Wild type bone chips in a FAK mutant injury site. (J) Mutant bone chips in a FAK mutant injury site. Arrows indicate multi-nucleated osteoclasts that attached to the transplanted bone chips. bm: bone marrow, c: cortex, is: injury site. Bar: 100 μm.