Results

Sam68 Is Expressed in the Developing Skeleton of Embryonic Mice
The Sam68 mRNA is known to be widely expressed [38], whereas its pattern of protein expression in vivo remains to be defined. Sections of paraffin-embedded wild-type E14.5 and E16.5 embryonic mice were immunostained with the well-characterized AD1 anti-Sam68 antibody, raised in rabbits against an immunizing peptide that corresponds to amino acids 330 to 348 of the mouse Sam68 protein [46]. Sam68 immunoreactivity was observed in the skeleton and soft tissues of developing wild-type mice at E14.5 and E16.5 (Figure 1). Intense staining was seen in the nucleus of cells in the developing brain, heart, and small intestine (Figure 1B), as well as in chondrocytes in the nasal septum and the glandular tissue adjacent to the nasal cartilage (Figure 1C, panels A–D), in the vertebra and intervertebral discs (panels E–H) and in the epiphysis (panels I–K) and metaphysis (panel L) of long bones. Nuclear staining was observed in proliferating chondrocytes and hypertrophic chondrocytes (Figure 1C, panel H), in osteoblasts (panel L; Dataset S1), and in osteoclasts (Dataset S1). No staining was observed with preimmune serum or when the antiserum was preadsorbed with the immunizing peptide (Figure 1C, panels B, F, and J). Taken together, these data demonstrate that Sam68 protein is selectively expressed in the developing mouse embryo, with particularly elevated expression in cartilage and bone.
Figure 1  Immunohistochemical Localization of Sam68 in Embryonic Mice
(A) Embryonic mice were removed from pregnant dams at E14.5 and E16.5, fixed in 4% paraformaldehyde, and embedded in paraffin. The entire embryo was immunostained with the AD1 anti-Sam68 antibody and counterstained with methyl green, and the image was captured at ×1.2 magnification.
(B) Embryonic soft tissues from the brain, heart and gut were stained with hematoxylin (left) and immunostained with anti-Sam68 antibody (right), and images were captured at ×20 magnification.
(C) Intense anti-Sam68 immunoreactivity was seen in chondrocytes in the nasal septum (panels A–D), in developing vertebra (panels E–H), and in the femoral epiphysis (panels I–K), as well as in diaphyseal osteoblasts (panel L). Adjacent sections were stained with hematoxylin and eosin (panels A, E, and I) or with antibody preadsorbed with the immunizing peptide (panels B, F, and J). Sam68 was localized primarily in the nucleus of cells in a variety of tissues but was also found occasionally in the cytoplasm. Magnification at source ×20, except for panels D, H, and L, which were ×40. Staining patterns are representative of three to five embryos.

Sam68 is Not Essential for Mouse Development
To define the physiologic role of Sam68, we generated Sam68-deficient mice by targeted disruption of exons 4 and 5 of the sam68 gene, which encode the functional region of the KH-type RNA binding domain (Figure 2A). The integrity of the targeted allele was verified by Southern blot analysis (Figure 2B) and by PCR of genomic DNA (unpublished data). Sam68 transcripts encoded by exons 1 to 5 were absent as evidenced by RT-PCR (Dataset S2), and Sam68−/− mice were devoid of Sam68 protein, as visualized by immunoblotting with anti-Sam68 AD1 and SC-333 antibodies or control normal rabbit serum or anti-Sam68 AD1 preabsorbed with peptide (Figure 2C). SC-333 is a rabbit anti-Sam68 antibody that was raised against the C-terminal 20 amino acids of Sam68 [47]. These data confirmed the generation of a mouse deficient in Sam68. The genotypes of offspring from heterozygote intercrosses exhibited a Mendelian segregation at E18.5 (Table 1). Despite the lack of visible deformity, many of the Sam68−/− pups died at birth of unknown causes. Sam68+/- mice were phenotypically normal and Sam68−/− pups that survived the perinatal period invariably lived to old age. Despite evidence that Sam68 mRNA is widely expressed and phosphorylated in mitosis [41,42], the Sam68−/− mice did not develop tumors and showed no immunologic or other major illnesses. Sam68−/− mice did, however, have difficulty breeding due to male infertility and the females rarely provided adequate care to their young.
Figure 2  Generation of Sam68-Deficient Mice
(A) The genomic organizations of the wild-type and targeted sam68 alleles after homologous recombination are depicted. The location of the DNA fragment used as a probe for the Southern blot analysis is shown, as well as the sizes of the two BglII fragments detected for wild-type and targeted sam68 alleles. The targeted allele replaces exon 4 and part of exon 5 of sam68 with a PGK-neomycin cassette.
(B) Southern-blot analysis of genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. DNA fragments corresponding to wild-type (4.5 kb) and the targeted (5.5 kb) alleles are illustrated.
(C) Western blot analysis of Sam68 expression. Protein extracts from wild-type, heterozygous, and homozygous cells subjected to immunoblot analyses using normal rabbit serum, anti-Sam68 AD1 antibody, the peptide antibody AD1 preabsorbed with the immunogenic peptide corresponding to amino acids 330–348 of mouse Sam68, anti-Sam68 Sc333 antibody that recognizes the C-terminal 20 amino acids of Sam68, and anti-actin antibodies as loading control. The migration of the molecular mass markers is known on the left in kDa.
Table 1  Progeny of Sam68 Heterozygote Breeding

Sam68 Deficiency Protects Mice from Age-Related Bone Loss
Src null animals are known to have bone metabolism defects [48], and since Sam68 is a substrate of Src, we decided to analyze the Sam68 mice for skeletal abnormalities. Cohorts of Sam68+/+ and Sam68−/− mice were euthanized at 4 and 12 months of age for skeletal phenotyping. To minimize differences in the bone phenotype that might arise secondary to gender or weight differences, we selected age-matched female mice for these analyses. The female mice demonstrated similar increases in body weight, although 4-month-old Sam68−/− mice weighed less than Sam68+/+ mice, and similar changes in bone lengths in the axial and appendicular skeleton between 4 and 12 months of age (Table 2). Faxitron radiography (Faxitron X-ray Corporation, Wheeling, Illinois, United States) (Figure 3A) and micro–computed tomography (CT) (Figure 3B) revealed significant cortical thinning (arrow) and a reduction in metaphyseal bone (asterisk) in the distal femora of 12-month-old Sam68+/+ mice compared with 4-month-old mice of either genotype and 12-month-old Sam68−/− mice. Similar reductions in trabecular bone were shown by Faxitron and micro-CT in the fifth lumbar vertebrae of the 12-month-old Sam68+/+ mice but not in the Sam68−/− mice (unpublished data). Total body bone mineral content (BMC), quantified with a Lunar PIXImus mouse densitometer (GE-Lunar, Madison, Wisconsin, United States), increased in both Sam68+/+ and Sam68−/− mice between 4 months and 12 months of age, but only reached significance in the Sam68−/− mice (Table 2). Similarly, a greater increase in BMC was seen in the femur and vertebra in the Sam68−/− mice. Bone mineral density (BMD) remained constant or decreased in the wild type mice but increased significantly in the total body and in the femoral and vertebral regions of interest in the Sam68−/− (Table 2). These data showed that Sam68−/− mice continued to thrive and accrue bone in the axial and appendicular skeleton for longer than 12 months. This was in contrast to the situation in age-matched, littermate controls in which a significant amount of bone was lost over the same timeframe.
Table 2  Morphology and Bone Mineral Density in 4- and 12-Month-Old Female Mice
Figure 3  Radiologic Assessment of the Femur of Young and Old Sam68+/+ and Sam68−/− Mice
(A) Mice were given a lethal dose of anesthetic at the indicated times, and contact radiographs of the distal femora were obtained on a Faxitron MX20 equipped with an FPX-2 Imaging system. Representative radiographs of the distal femur of Sam68+/+ (+/+) and Sam68−/− (−/−) mice revealed comparable radiopacity at 4 months (left). At 12 months (right), cortical thinning (arrow) and radiolucency (asterisk) were apparent in the distal femur of +/+ mice but not −/− mice.
(B) Bones were dissected free of soft tissue and fixed overnight in 4% paraformaldehyde before scanning on a Skyscan 1072 static instrument equipped with 3D Creator analytical software. Representative three-dimensional re-constructions and two-dimensional cross-sectional scans demonstrated similar architecture in the distal femur of Sam68+/+ (+/+) and Sam68−/− (−/−) mice. In keeping with the results from Faxitron x-ray, trabecular bone (asterisk) and cortical thickness (arrow) were reduced in the femur of 12-month-old +/+ mice compared with all other groups. The images are representative of those from five to seven animals in each group.

Three-Dimensional Architecture of Bone Is Preserved in Aged Sam68−/− Mice
Bone loss and compromised architecture are characteristic features of the skeletons of aged C57BL/6 mice and resemble the clinical features of age-related bone loss in humans that can predispose an individual to fracture. Quantification of the micro-CT data shown in Figure 3B confirmed the reduction in bone volume compared with tissue volume (BV/TV; Figure 4A, left) in the 12-month-old Sam68+/+ mice (hatched bars) compared with 4-month-old mice of both genotypes (solid bars) and 12-month-old Sam68−/− mice (stippled bars). This was associated with a significant increase (p < 0.01; denoted by the asterisks) in the structure model index, which measures the ratio of plate-like to rod-like structures (Figure 4A, right). A quantifiable increase in the mean trabecular separation (Tb.Sp) (unpublished data) in 12-month-old Sam68+/+ mice was due to an increase in the percentage of spaces falling in the range of 350 to 700 μm (Figure 4B, red hatched line). Trabecular thickness remained constant among the different groups of mice (unpublished data). In effect, this meant that there were fewer trabeculae, rather than equivalent numbers of thin trabeculae, in the 12-month-old Sam68+/+ mice compared with any of the other groups of mice.
Figure 4  Quantitative Micro-CT of Trabecular Bone Composition and Architecture
(A) Bone volume/tissue volume (BV/TV) and structure model index (SMI) were calculated on the femur and fourth lumbar vertebra of six or seven mice in each group using 3D Creator software supplied with the Skyscan instrument. Results expressed as the mean ± SD showed significant differences (p < 0.01) between 4-month-old Sam68+/+ mice (solid black) and 12-month-old Sam68+/+ mice (hatched black) but not between 4-month-old Sam68−/− mice (solid white) and 12-month-old Sam68−/− mice (stippled white).
(B) The distance between trabeculae was reflected in a shift to the right of the distribution curves for 12-month-old Sam68+/+. Solid black = 4-month-old Sam68+/+; hatched black = 12-month-old Sam68+/+; solid red = 4-month-old Sam68−/−; stippled red = 12-month-old Sam68−/−. The asterisks denote p < 0.01.

Bone Remodeling Is Preserved in Aged Sam68−/− Mice
To further define the mechanisms involved in the preservation of bone mass in aged Sam68−/− mice, we prepared sections from plastic-embedded femora and tibia to evaluate osteoblast and osteoclast activity (Figure 5). In situ enzyme histochemical staining for alkaline phosphatase (ALP; brown stain) activity was used as a biomarker for osteoblasts and tartrate-resistant ALP (tartrate-resistant acid phosphatase [TRAP]; red stain) activity as a marker for osteoclasts. Little difference was seen between Sam68+/+ (Figure 5A and 5B) and Sam68−/− (Figure 5C and 5D) mice at 4 months of age. ALP- and TRAP-positive cells were reduced in the 12-month-old Sam68+/+ mice (Figure 5E and 5F) and remained unchanged in 12-month-old Sam68−/− mice (Figure 5G and 5H). The reduction in both osteoblast and osteoclast activity in the 12-month-old Sam68+/+ mice argued against bone being lost primarily due to a relative increase in osteoclast over osteoblast activity, as seen in high turnover disease [49].
Figure 5  Histologic Analysis of Undecalcified Bone from Sam68+/+ and Sam68−/− Mice
Sections of tibia fixed in 4% paraformaldehyde and embedded in plastic were stained for ALP (A–C, E–G) activity to identify osteoblasts or for TRAP (B–D, F–H) activity to identify osteoclasts. Staining patterns were similar in 4-month-old Sam68+/+ (A and B), 4-month-old Sam68−/− (C and D), and 12-month-old Sam68−/− (G and H) mice compared with 12-month-old Sam68+/+ mice (E and F). Magnification at source, left panels ×10 and right panels ×40. Micrographs are representative of those taken from five to seven sections in each group of animals. Histomorphometric analyses [50] of the long bones (Table 3) corroborated the radiologic evidence of age-related bone loss. Bone volume (BV/TV), newly formed osteoid (OV/TV), and mineral apposition rate (MAR) were all significantly reduced in 12-month-old Sam68+/+ mice compared with 4-month-old Sam68+/+ mice. These reductions were associated with a significant increase in marrow fat (FV/TV) and decreased numbers of osteoblasts (nOB/TV) and osteoclasts (nOC/TV) per tissue volume (Table 3). These results were in contrast to those of the 12-month-old Sam68−/− mice in which all histomorphometric parameters, including marrow fat, resembled those of young mice of either genotype (Table 3). When cells were expressed as a function of the bone perimeter (nOB/BP, nOC/BP), there were no statistical differences and the ratio of osteoblasts to osteoclasts was similar in all groups of mice (Table 3).
Table 3  Histomorphometric Analysis of Long Bones from 4- and 12-Month-Old Mice Circulating levels of serum C-telopeptide (sCTX; Roche Diagnostics, Mannheim, Germany) and ALP showed little difference among the groups, except for a small decrease in 12-month-old Sam68−/− mice and (Dataset S3). Serum estrogen was significantly decreased in all 12-month-old mice regardless of genotype with a reciprocal increase in interleukin-6 levels (Dataset S3). Given the involvement of leptin in regulating body and bone mass, serum leptin was measured in Sam68+/+ and Sam68−/− mice. Twelve-month-old Sam68−/− mice had significantly lower levels than young and old Sam68+/+ mice and young Sam68−/− mice (Dataset S3). Taken together, these observations suggested that preservation of bone mass in the Sam68−/− mice was not due primarily to altered estrogen status but could have been influenced by differences in circulating leptin levels.

Osteoblast, but Not Osteoclast, Activity Is Altered in Sam68−/− Mice Ex Vivo
Maintenance of bone mass during the adult remodeling cycle is dependent on the coupling of osteoblast to osteoclast activity, such that there is no net gain or loss of bone [49]. To further explore the age-related advantage of Sam68−/− mice with respect to bone preservation, we examined the functional activity of Sam68−/− osteoblasts and osteoclasts ex vivo (Figure 6A and 6B). Cultures of bone marrow stromal cells harvested from 4-week-old Sam68−/− mice and maintained for 18 days in osteoblast differentiation medium demonstrated more intense staining for ALP at 6 days (unpublished data) and 18 days (Figure 6A), and more mineralized nodules at 18 days, than the Sam68+/+ mice, even though similar amounts of fibroblast colony-forming units (CFU-F) were observed (Figure 6A and unpublished data). RT-PCR analysis of molecular markers of osteoblast differentiation (Dataset S2) revealed similar increases over time in RNA from Sam68+/+ and Sam68−/− mice, although type I collagen appeared to be up-regulated at both timepoints in the Sam68−/− mice. Short-term cultures of mature osteoclasts released from the crushed long bones of Sam68+/+ and Sam68−/− mice stained equally well for TRAP and excavated approximately equal numbers of pits of equal size in dentin slices, as visualized by scanning electron microscopy (Figure 6B). These findings suggest that the osteoclasts may not be the primary defect in Sam68−/− mice, as they are in Src null mice [48].
Figure 6  Ex Vivo Activity of Sam68+/+ and Sam68−/− Osteoblasts and Osteoclasts
Marrow stromal cells were isolated from the long bones of juvenile mice and maintained under conditions that promote osteoblast differentiation.
(A) Cultures were fixed in 4% paraformaldehyde after 6 or 18 days and stained in situ for ALP activity and with silver nitrate (von Kossa) to detect mineralized nodules. Sam68−/− cultures stained more intensely for ALP at early and late time points and produced significantly more mineralized nodules after 18 days. Asterisks represent p < 0.01.
(B) Primary osteoclasts were isolated from the crushed long bones of the same mice and plated on glass coverslips or on dentin slices to quantify numbers and activity, respectively. Osteoclasts were identified as cells with three or more nuclei that stained positive for TRAP activity (upper) and excavated pits in dentin slices, as demonstrated by SEM (lower, bar = 20 μm). No statistical differences were observed either in the number of TRAP-positive cells or in their resorptive activity.

The Absence of Sam68 Prevents Adipocyte Differentiation and Promotes Osteoblast Differentiation
It is well recognized that bone marrow stromal cells give rise to both osteoblasts and adipocytes and that age-related bone loss is accompanied by an increase in differentiation down the adipocyte lineage [22]. Therefore, the loss of Sam68 could influence the bone marrow stromal cells to differentiate along the osteogenic versus the adipogenic pathway. Alternatively, the loss of Sam68 could indirectly regulate bone mass as a general disturbance of neuroendocrine control as was shown when leptin and the sympathetic nervous system axis were shown to negatively regulate bone mass [8]. To confirm a role for Sam68 in the regulation of adipocyte differentiation, we isolated primary MEFs from 14.5-day-old Sam68+/+ and Sam68−/− embryos. The primary Sam68−/− MEFs were differentiated into adipocytes in vitro in culture medium containing 5 μM pioglitazone to induce adipogenesis. Cells at days 0, 4, 6, and 12 were stained with Oil red O to monitor adipogenesis. Adipogenesis was more pronounced in the wild-type MEFs cultures than in the Sam68−/− MEFs, consistent with the positive role of endogenous Sam68 in adipocyte differentiation (Figure 7). The expression of key transcription factors including the PPARγ and KLF5 was impaired in Sam68−/− differentiated MEFs compared with Sam68+/+ MEFs, consistent with impaired adipogenesis in the absence of Sam68 (Figure 7). These data, together with data confirming a lean phenotype in Sam68−/− mice (N. Torabi and S. Richard, unpublished data), support the hypothesis that Sam68 modulates the differentiation of mesenchymal cells.
Figure 7  Ex Vivo Adipogenesis Analysis of Sam68−/− Mouse Embryonic Fibroblasts
MEFs were isolated from mouse embryos at embryonic day 14.5. Equal number of MEFs from Sam68+/+ and Sam68−/− was plated on glass cover slips in 24 well-plates. Adipocyte differentiation was carried out at indicated times by the addition of complete media containing the pioglitazone.
(A) Cultures were fixed in 4% paraformaldehyde and stained with Oil Red O to detect the fat droplets stored in adipocytes and photographed (top). The cell images were magnified ×10 and ×20 as indicated.
(B) RT-PCR was carried out on total cellular RNA isolated after differentiation of the MEFs for day 0, 2, 4, 6, and 12. The DNA fragments were visualized on agarose gels stained with ethidium bromide. The expression of adipogenic markers C/EBPβ, C/EBPδ, PPARα, and KLF5 was examined as well as the expression of controls including Sam68, β-actin, and GAPDH. To further examine this phenotype in a cell autonomous system, we chose the embryonic mesenchymal multipotential progenitor cells C3H10T1/2. The addition of BMP-2 induced osteoblast differentiation, as evidenced by an approximately 400-fold increase in expression of the osteocalcin (OCN) gene [51]. Populations of C3H10T1/2 cells stably transfected with either pSuper-retro (control) and pSuper-retro harboring a short hairpin against Sam68 (Sam68sh) were selected with puromycin. The expression of Sam68 was reduced by approximately 80% as evidenced by immunoblot analyses using β-actin as a loading control (Figure 8A). Osteoblast differentiation was induced in cultures expressing pSuper-retro or pSuper-retro Sam68sh by addition of BMP-2 to the culture medium. . The expression of OCN and β-actin mRNAs was examined by semiquantitative RT-PCR and the Sam68sh-expressing cells displayed a more pronounced osteoblast phenotype compared with control cells, as assessed by the expression of OCN (Figure 8B).
Figure 8  Enhanced Osteogenic Differentiation of the C3HT101/2 Embryonic Cell Line Depleted of Endogenous Sam68
(A) C3HT101/2 cells transfected with an empty vector (pSuper-retro) or a vector containing an shRNA (Sam68 shRNA) were selected with puromycin, and knockdown populations depleted of Sam68 were identified. The reduction in Sam68 protein was analyzed by immunoblotting with anti-Sam68 (AD1) antibody and anti–β-actin antibodies as loading controls.
(B) Osteogenic differentiation was carried out with conditioned medium containing BMP-2 for the indicated times. To assess the level of osteogenic differentiation in these cells, expression of late osteoblast marker, osteocalcin (OCN), was analyzed by RT-PCR and compared with β-actin and GAPDH controls. The DNA fragments were visualized by agarose gel stained with ethidium bromide. Given the apparent enhancement of mineralized nodule formation by Sam68−/− bone marrow stromal cells ex vivo and the phenotype observed with short hairpin RNA (shRNA)-treated C3H10T1/2, we stained sections of bone from 4- and 12-month-old mice for evidence of changes in marrow adiposity. Figure 9 shows sections of undecalcified bone from 4- and 12-month-old Sam68+/+ and Sam68−/− mice stained with von Kossa and toluidine blue to show mineralized tissue (Figure 9A, black) and marrow adipocytes (Figure 9B, white) and left unstained to show fluorochrome labeling of the mineralization fronts (Figure 9C). Twelve-month-old Sam68+/+ mice showed a noticeable decrease in trabecular bone (Figure 9A), which was associated with a significant increase in marrow adiposity (Figure 9B) and with the two fluorochrome labels superimposed upon one another (Figure 9C). In contrast, the bones of 12-month-old Sam68−/− mice appeared similar to those of the 4-month-old mice of either genotype. These data demonstrate that Sam68 regulates the differentiation of bone marrow mesenchymal cells to promote adipocyte differentiation and inhibit osteoblast differentiation in aging bone.
Figure 9  Old Sam68−/− Mice Are Protected from the Development of Fatty Bone Marrow
Sections of undecalcified bone were stained with von Kossa and toluidine blue and images captured at original magnifications of ×2 (A), ×40 (B and C) to evaluate mineralized tissue (A, black), marrow adipocytes (B, white), and the mineralization fronts (C, yellow and green). The 12-month-old Sam68+/+ bone demonstrated a significant reduction in bone (A) and increase in marrow adipocytes (B) and a decrease in the distance between two consecutive fluorochrome labels (C). Magnification at source was ×40. Micrographs are representative of four to six screened in each group of animals.