Decreased c-Src expression enhances osteoblast differentiation and bone formation

Abstract

c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src(-/-) relative to Src(+/+) mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by approximately 60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src(-/-) osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.

Figure 1

Histomorphometric analysis of tibiae from 3- and 10-wk-old Src^+/+ and Src^−/− mice. Samples from 3 and 10-wk-old Src^+/+ and Src^−/− mice were fixed, embedded in methylmethacrylate, and evaluated for histomorphometric analysis as described in the Materials and Methods. All values are mean ± SEM from at least three animals for each category. Src^+/+; Src^−/−; *P < 0.05, **P < 0.01 and ***P < 0.001 versus Src^+/+ mice.

Figure 2

Src-deficient osteoblast phenotype. Primary calvarial osteoblasts were harvested from B6, 129^tm/sor mice, as described in Materials and Methods, and cultured for detection of ALP activity and nodule mineralization. (a) 90% confluent osteoblasts were cultured for 1 wk, fixed, and stained histochemically to detect ALP activity using the Sigma kit n. 85. ALP-positive cells were counted and converted into a percentage versus the total number of cells. Data are the mean ± SEM of three independent experiments. ***P < 0.001 versus Src^+/+. (b) 90% confluent osteoblasts were cultured for 3 wk in the presence of ascorbic acid and β-glycerophosphate, as described in Materials and Methods. Cultures were then fixed and mineralized (dark) nodules were detected by von Kossa staining. Quantitative analysis was performed by scanning densitometry, as described in Materials and Methods, and data from three independent experiments were expressed as arbitrary densitometric units (mean ± SEM). **P < 0.01 versus Src^+/+.

Figure 2

Src-deficient osteoblast phenotype. Primary calvarial osteoblasts were harvested from B6, 129^tm/sor mice, as described in Materials and Methods, and cultured for detection of ALP activity and nodule mineralization. (a) 90% confluent osteoblasts were cultured for 1 wk, fixed, and stained histochemically to detect ALP activity using the Sigma kit n. 85. ALP-positive cells were counted and converted into a percentage versus the total number of cells. Data are the mean ± SEM of three independent experiments. ***P < 0.001 versus Src^+/+. (b) 90% confluent osteoblasts were cultured for 3 wk in the presence of ascorbic acid and β-glycerophosphate, as described in Materials and Methods. Cultures were then fixed and mineralized (dark) nodules were detected by von Kossa staining. Quantitative analysis was performed by scanning densitometry, as described in Materials and Methods, and data from three independent experiments were expressed as arbitrary densitometric units (mean ± SEM). **P < 0.01 versus Src^+/+.

Figure 3

Osteoblast phenotype in Src^+/+ and Src^−/− immortalized cells. (a) Src protein expression in immortalized osteoblasts. Confluent monolayers were lysed and 60 μg of total cell protein was electrophoresed in a 10% SDS-PAGE, blotted to nitrocellulose filter paper, and probed with Src pAb (diluted 1:250) at 4°C overnight, followed by the HRP-conjugated secondary antibody (diluted 1:5,000) at 37°C for 1 h. The filter was stripped and reprobed with anti-actin pAb (diluted 1:250). Bands were detected by ECL. This evaluation was repeated three times with similar results. (b) 90% confluent Src^+/+ and Src^−/− immortalized osteoblasts were cultured for the indicated times and subjected to determination of ALP, as described in the legend to Fig. 2. Cultures were treated with or without 10⁻⁷ M dexamethasone (Dex) for the entire time frame. Positive nodules are evident as dark spots. This experiment was repeated three times with similar results. (c) 90% confluent Src^+/+ and Src^−/− immortalized osteoblasts were cultured for 3 wk in the presence of ascorbic acid and β-glycerophosphate, as described in the Materials and Methods and with or without 10⁻⁷ M Dex for the entire time frame. Cultures were then fixed and subjected to von Kossa staining for the detection of nodule mineralization (dark areas). Quantitative analysis was performed by scanning densitometry, as described in the Materials and Methods, and data from three independent experiments were expressed as arbitrary densitometric units (mean ± SEM). ***P < 0.001 versus Src^+/+; ND, non detectable.

Figure 4

Immunoblotting analysis of Src expression in calvarial osteoblasts. Confluent calvarial osteoblasts from CD1 mice were treated with 1 μM AS-src or S-src for 24 h. Cells were lysed, subjected to SDS-PAGE, and immunoblotted for Src and actin, as described in the legend to Fig. 3. Bands were detected by ECL. Band densitometric analysis was performed as described in the Materials and Methods. The Src/actin ratio for each treatment was computed. Similar results were observed in three independent experiments performed with three different osteoblast preparations. Statistical significance between AS-src and S-src was <0.05. C, control; S, S-src ODN; AS, AS-src ODN.

Figure 5

Osteoblast proliferation. (a) To assess osteoblast proliferation, 70% confluent cultures were treated for 48 h with 1 μM AS-src (AS), 1 μM S-src (S), or left untreated (C). 1 μCi/ml of [³H]thymidine was added for the last 4 h, then the cells were lysed, TCA-precipitated, and the radioactivity was measured by a β-spectrophotometer. Counts per minutes were then converted to a percentage of control and expressed as average ± SEM of three independent experiments. Statistical significance was <0.05 (*) versus S-src. (b) Primary osteoblasts and (c) immortalized Src^+/+ and Src^−/− osteoblasts were cultured to 70% confluence, 1 μCi/ml of [³H]thymidine was added for 4 h, and cells were processed as described in the legend to a. Data are the mean ± SEM of three independent experiment. Statistical significance was <0.01 (**) versus Src^+/+.

Figure 6

ALP activity. (a) 90% confluent calvarial osteoblasts from CD1 mice were treated for 3 d with 1 μM AS-src, 1 μM S-src, or left untreated. Cells were fixed, stained histochemically for ALP activity using the Sigma kit n. 85, and observed by conventional light microscopy. (b) The number of ALP-positive cells was counted and expressed as mean percentage ± SEM of control. Data are from three independent experiments. *P < 0.05 versus S-src. (c) Osteoblasts were lysed and ALP activity was evaluated biochemically using the Sigma kit n. 104-LS and p-nitrophenyl phosphate were used as substrate. Activity, normalized versus protein content, was then converted as a percentage of control. Data are mean ± SEM of three independent experiments, *P < 0.05 versus S-src. C, control; S, S-src ODN; AS, AS-src ODN.

Figure 6

ALP activity. (a) 90% confluent calvarial osteoblasts from CD1 mice were treated for 3 d with 1 μM AS-src, 1 μM S-src, or left untreated. Cells were fixed, stained histochemically for ALP activity using the Sigma kit n. 85, and observed by conventional light microscopy. (b) The number of ALP-positive cells was counted and expressed as mean percentage ± SEM of control. Data are from three independent experiments. *P < 0.05 versus S-src. (c) Osteoblasts were lysed and ALP activity was evaluated biochemically using the Sigma kit n. 104-LS and p-nitrophenyl phosphate were used as substrate. Activity, normalized versus protein content, was then converted as a percentage of control. Data are mean ± SEM of three independent experiments, *P < 0.05 versus S-src. C, control; S, S-src ODN; AS, AS-src ODN.

Figure 7

Nodule mineralization. Osteoblasts were plated in 24-well multiplates, grown to 90% confluence, and incubated for 3 wk with ascorbic acid and β-glycerophosphate, as described in the Materials and Methods, with the addition of 1 μM AS-src or S-src, with or without 10⁻⁷ M dexamethasone (Dex). Cultures were fixed and mineralization (dark areas) was evidenced by the von Kossa reaction. Quantitative analysis was performed by scanning densitometry, as described in the Materials and Methods, and data from three independent experiments are expressed as arbitrary densitometric units (mean ± SEM). *P < 0.05 versus S-src. C, control; S, S-src ODN; AS, AS-src ODN.

Figure 9

Transcriptional regulation of osteoblast genes in primary Src^+/+ and Src^−/− calvarial cells. Src^+/+ and Src^−/− primary osteoblasts were grown to confluence, then RNA was extracted. 1 μg RNA was reverse-transcribed and the equivalent of 0.1 μg was subjected to PCR using primer pairs and conditions as described in the legend to Table . Densitometric analysis was performed as described in the Materials and Methods, then the density ratio between the gene being analyzed and the constitutive gene GAPDH was computed. Similar results were observed in three independent experiments, with P < 0.05 for Src^−/− versus Src^+/+.

Figure 8

Transcriptional regulation of osteoblast genes. 90% confluent osteoblasts were incubated for 3 d with 1 μM AS-src, 1 μM S-src, or left untreated, and RNA was extracted. 1 μg RNA was reverse-transcribed and the equivalent of 0.1 μg was subjected to PCR using primer pairs and conditions as described in the legend to Table . Densitometric analysis was performed as described in Materials and Methods, and then the density ratio between the gene being analyzed and the constitutive gene GAPDH was computed. Similar results were observed in three independent experiments, with P < 0.05 for AS-src treated versus S-src. C, control; S, S-src ODN; AS, AS-src ODN.

Figure 10

Transcriptional regulation of osteoblast genes in immortalized cells. Src^+/+ and Src^−/− immortalized osteoblasts were grown to confluence, then RNA was extracted. 1 μg RNA was reverse-transcribed and the equivalent of 0.1 μg was subjected to PCR using primer pairs and conditions as described in the legend to Table . Densitometric analysis was performed as described in the Materials and Methods, and then the density ratio between the gene being analyzed and the constitutive gene GAPDH was computed. Similar results were observed in three independent experiments, with P < 0.05 for Src^−/− versus Src^+/+.

Figure 11

Expression of osteopontin. RNA or proteins were extracted from 90% confluent osteoblasts treated for 3 d with 1 μM AS-src, 1 μM S-src, or left untreated, or from Src^+/+ and Src^−/− primary and immortalized osteoblasts. (a) RT-PCR was performed using the osteopontin (OPN) primer pairs and conditions as described in the legend to Table . Densitometric analysis was performed as described in the Materials and Methods, then the density ratio between the gene being analyzed and the constitutive gene GAPDH was computed. Similar results were observed in three independent experiments. Differences are not statistically significant. (b) 25 μg protein for each sample was electrophoresed in a 10% SDS-PAGE, blotted to nitrocellulose filter paper, and probed with L-123 anti-OPN pAb (diluted 1:250) at 4°C overnight, followed by the HRP-conjugated secondary antibody (diluted 1:5,000) at 37°C for 1 h. Filters were stripped and reprobed with anti-actin pAb (diluted 1:250). Bands were detected by ECL and densitometric analysis was performed as described in the Materials and Methods, and then the OPN/actin density ratio was computed. Similar results were observed in three independent experiments. Differences are not statistically significant. C, control; S, S-src ODN; AS, AS-src ODN.