|Home | About | Journals | Submit | Contact Us | Français|
The ubiquitously expressed calpains-1 and -2 belong to a family of calcium-dependent intracellular cysteine proteases. Both calpains are heterodimers consisting of a large catalytic subunit and a small regulatory subunit encoded by the gene Capn4. Ablation of the calpain small subunit eliminates calpain activity and leads to embryonic lethality. We previously created osteoblast-specific Capn4 knockout mice to investigate a physiological role for the calpain small subunit in cells of the osteoblast lineage. Deletion of Capn4 reduced trabecular and cortical bone mainly due to impaired proliferation and differentiation of cells of the osteoblast lineage. To further investigate an underlying mechanism by which osteoblast-specific Capn4 knockout mice develop an osteoporotic bone phenotype, we established osteoblastic cell lines stably expressing either control or Capn4 RNA interference (RNAi) for this study. Capn4-knockdown cells showed reduced cell proliferation, accumulation of total and phosphorylated cyclin-dependent kinase inhibitor 1B (p27Kip1) on Serine (S) 10, reduced phosphorylation of retinoblastoma protein (Rb) on Threonine (T) 821. Moreover, ablation of Capn4 increased 27Kip1 mRNA levels likely due to stabilized binding of Akt to protein phosphatase 2A (PP2A), which presumably results in reduced phosphorylation of Akt on S473 and forkhead Box O (FoxO) 3A on T32. Collectively, calpain regulates cell proliferative function by modulating both transcription and degradation of p27Kip1 in osteoblasts. In conclusion, calpain is a critical modulator for regulation of p27Kip1 in cells of the osteoblast lineage.
Osteoporosis is a bone disease that leads to an increased risk of bone fracture, thus may significantly affect life expectancy and quality of life. Osteoporotic bone is characterized by low bone mineral density, disrupted bone microarchitecture, and abnormal amount and composition of bone matrix proteins .
Calpains are calcium-dependent intracellular cysteine proteases. Both calpains-1 and -2 consist of an 80-kDa large catalytic subunit encoded by the genes Capn1 and Capn2, respectively, and a 28-kDa common small regulatory subunit encoded by the gene Capn4 . Deletion of the calpain small subunit eliminates calpain activity and leads to embryonic lethality, suggesting an essential role of Capn4 during embryonic development .
Several lines of evidence have suggested that calpain plays a crucial role in parathyroid hormone (PTH)-mediated cellular functions in osteoblasts; PTH induces osteoblastic retraction likely caused by a calpain-dependent proteolytic modification of cytoskeletal organization [4–7]. PTH also stimulates activities of calpains-1 and -2 [5, 6] and pretreatment of MC3T3-E1 osteoblastic cells with calpain inhibitors blocks PTH-stimulated cell proliferation and differentiation [7, 8].
We previously showed that the calpain small subunit binds to the intracellular C-terminal tail of the receptor for PTH and PTH-related peptide (PTHrP) (PTH1R), and critically modulates ligand-mediated PTH1R signaling . To investigate a role of the calpain small subunit in cells of the osteoblast lineage in vivo, we then generated osteoblast-specific Capn4 knockout mice. Deletion of Capn4 exhibited reduced trabecular and cortical bone mainly due to reduced proliferation and differentiation of cells of the osteoblast lineage . However, we failed to provide the underlying molecular mechanism by which deletion of the calpain small subunit modulates osteoblast function. In our more recent study using chondrocyte-specific Capn4 knockout mice, we found that deletion of Capn4 reduces cell proliferation, at least in part, through accumulation of p27Kip1 protein in cells of the chondrocyte lineage . Therefore, to further test our hypothesis that calpain also critically modulates p27Kip1 in cells of the osteoblast lineage, we established osteoblast cell lines stably expressing either control or Capn4 RNAi and examined whether and how knockdown of Capn4 modulates p27Kip1 protein levels in cells of the osteoblast lineage in vitro in this study.
Mouse osteoblastic cells, MC3T3 Subclone4 (MC4) (ATCC, Manassas, VA), stably expressing either control or Capn4 microRNAs are established as we described previously . Control and Capn4 microRNAs were commercially available from Invitrogen Corp. (Carlsbad, CA). Four monoclonal cell lines each were established, and knockdown of the calpain small subunit was assessed by calpain activity assay as we described previously . MC4 stable cell lines were cultured in α minimal essential medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and 1% penicillin-streptomycin (Invitrogen). Mouse p27Kip1 siRNAs (Invitrogen) were also used to knockdown p27Kip1 protein as we described previously . Antibodies against PP2A, total (t)-Akt, t-FoxO3A, phosphorylated (p)-Akt (S473), p-FoxO3A (T32) (Cell Signaling Technology Inc., Danvers, MA), cyclin D, cyclin E, p27Kip1, cyclin-dependent kinase 2 (cdk2) and 4 (cdk4) (Santa Cruz Biotechnology, Santa Cruz, CA), p-retinoblastoma protein (Rb) (T821) (Invitrogen), and p-p27Kip1 (S10) (Abcam Inc., Cambridge, MA) were purchased.
To assess apoptosis of established cell lines, cells were stained with Annexin V-phycoerythrin and 7-amino actinomycin D using Guava PCA Nexin kit and analyzed by Guava Personal Cytometer (Guava Technology Inc., Hayward, CA) as described previously [10, 11].
Cell cycle analysis was performed using flow cytometric analysis as we described previously . MC4 cells stably expressing control or Capn4 microRNA were serum starved (1% FBS) for 2 days and then stimulated by serum replacement (10% FBS) for 10 h. Cells were labeled with 10 μM bromodeoxyuridine (BrdU) for the last 1 h, harvested, and stained with anti-BrdU fluorescein isothiocyanate antibody for BrdU and propidium iodine for DNA as recommended by the manufacturer (Becton Dickinson and Company [BD], San Jose, CA). A total of 10,000 events were analyzed for each sample. The percentage of cells in G1, S, and G2/M phases of the cell cycle was determined by use of a BD LSRII analyzer, followed by analysis using FlowJo (Tree Star Inc., Ashland, OR) software.
Calpain activity assay was performed as we described previously . In brief, cell lysates (10 μg/sample) were incubated with 200 μM calpain substrate, N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Bachem, Torrance, CA) in a buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 3mM Ca2+ at 37°C for 20 min. Fluorescence was measured at 460 nm with excitation at 355 nm by using a Vector3 plate reader, and data were analyzed using Wallac software (PerkinElmer Life and Analytical Science Inc., Waltham, MA). 7-Amino-4-methylcoumarine (Bachem) was used as a standard.
Real-time qRT-PCR was performed as described previously . Samples were run in duplicate and the results were normalized to GAPDH expression. Primer sequences for GAPDH, Cyclin D1, Cyclin E1, and p27Kip1 were as follows: GAPDH, (forward, 5’-AACTACATGGTC TACATGTTCCA-3’ and reverse 5’-CCATTCTCGGCCTTGACTGT-3’); Cyclin D1, (forward, 5’-GGGGACAACTCTTAAGTCTCAC-3’ and reverse, 5’-CCCAATAAAAGACCAATCTCTC-3’); Cyclin E1 (forward, 5’-CACAACATCCAGACCCACAC-3’ and reverse, 5’-ATGGCAGGTTTGGTCATTCT-3’); and p27Kip1 (forward, 5’-TAGAGATGGCACAGGGTTCC-3’ and reverse, 5’-CCCATCCAATTCGACAACTT-3’).
Western blot analysis was performed as described previously  with antibodies against the following proteins: cyclin D, cyclin E, cdk2, cdk4, p27Kip1, p-retinoblastoma protein (T821), p-p27Kip1 (S10), t-Akt, p-Akt (S473), t-FoxO3A, and p-FoxO3A (T32). In some experiments, blots were incubated in Restore Western blot stripping buffer (Thermo Scientific, Rockford, IL) and reprobed with other primary antibodies. The intensity of the selected protein bands was semi-quantified using FluorChem SP (Alpha Innotech Corp., San Leandro, CA). The relative density of p-Akt (S473)/t-Akt and p-FoxO3A(T32)/t-FoxO3A at time 0 was set as 1 in control and Capn4-kdn cells, respectively. A rate constant for dephosphorylation of p-Akt (S473) and p-FoxO3A (T32) was calculated using one phase exponential decay curve fit program of Prism 4 Software (GraphPad Software Inc., La Jolla, CA).
For immunoprecipitation analysis, MC4 cells stably expressing either control or Capn4 RNAi were harvested, and cell lysates were precleared with 0.5 μg/ml normal mouse IgG agarose (Santa Cruz) for 2 h at 4°C with agitation. One milligram of protein of the supernatant was incubated with 2 μg of primary antibodies to Akt for 1 h at 4°C and then with 40 μl of protein A/G agarose (Santa Cruz) overnight at 4°C. Samples were washed, dissolved in sample buffer, and resolved on a 10% SDS gel followed by Western blot analysis using anti-PP2A antibody. Some samples were also used for phosphatase activity assay.
PP2A phosphatase activity was measured using Immunoprecipitation Phosphatase Assay Kit (Millipore) with modification using 2 nM okadaic acid, a PP2A inhibitor .
Data were calculated from three and more independent experiments and expressed as the mean±SE. Statistical analysis was performed using the unpaired Student’s T-test or analysis of variance. p values less than 0.05 were accepted as significant.
To test a role of Capn4 in cell proliferation of the osteoblast lineage, we created MC3T3 Subclone4 (MC4) cell lines stably expressing either control or Capn4 microRNAs. The average calpain activities of established cells lines, MC4-Capn4 microRNA 1 and 2, were 50% and 35% vs. MC4-Control microRNA (control) lines 107±2% (n=4), respectively. We used MC4-Capn4 microRNA 2 (Capn4-knockdown [kdn]) cell line, whose calpain activity was reduced to approximately 35% of that of control cells, in most of our experiments.
Stable deletion of Capn4 in osteoblasts showed impaired cell growth assessed by cell number (Fig. 1a). Proliferation of control and Capn4-kdn cells was assessed by BrdU-incorporation followed by flow cytometry; the number of BrdU-positive cells was reduced by approximately 30% in Capn4-kdn cells compared with control cells (% cells in S phase; control 24±3 % vs. Capn4-kdn 16±2 %). No difference was observed in apoptosis between control and Capn4-kdn osteoblastic cells (% of apoptotic cells; control, 8.8±0.3% vs. Capn4-kdn, 7.1±1.1%). Collectively, knockdown of Capn4 impairs cell proliferation of the osteoblast lineage as we reported previously .
To identify a mechanism by which knockdown of Capn4 reduces cell proliferation of the osteoblast lineage, we next examined mRNA expression of cell cycle proteins, cyclin D1 and cyclin E1. Expression of cyclinD1 mRNA was significantly reduced in Capn4-kdn MC4 osteoblastic cells (Fig. 1b); this result was consistent with our reports that mRNA expression of c-fos, a downstream target of PTH and a positive regulator of cyclin D transcription, is impaired by ablation of Capn4 in cells of the osteoblast  and chondrocyte lineages . Levels of various cell cycle proteins were next examined. Despite reduced cyclin D1 mRNA levels (Fig. 1b), increased protein levels of p27Kip1 (Fig. 1c, 1d, and 1e) and cyclin D (Fig. 1d and 1e) were detected by Western blot analysis. Notably, p27Kip1 and cyclin D are calpain substrates [13, 14]. We also observed reduced phosphorylation of Rb on T821 in Capn4-kdn cells compared with control cells (Fig. 1d and 1e). There were no significant differences in protein levels of cdk4 (Fig. 1d and 1e), cyclin E, cdk 2, and p21Cip1 (data not shown) between control and Capn4-kdn osteoblasts. Thus, these results suggest that, despite increased cyclin D protein levels, increased p27Kip1 effectively blocks a cyclin D-cdk4 complex and reduces phosphorylation of Rb, resulting in impaired cell cycle transition at G1/S as we described previously in cells of the chondrocyte lineage .
Moreover, phosphorylation of p27Kip1 on S10 was previously shown by others and us to be associated with calpain-mediated degradation of p27Kip1; ablation of Capn4 impairs phosphorylation of p27Kip1 on S10  and removal of p-S10 p27Kip1 through a calpain-mediated degradation pathway . Deletion of Capn4 in osteoblastic cells also showed approximately twofold increased levels of p-S10 p27Kip1 protein (Fig. 1d and 1e), suggesting impaired calpain-mediated degradation of p-S10 p27Kip1 protein in osteoblasts. Next, we tested whether additional knockdown of p27Kip1 rescues an impaired cell growth phenotype of cells of the osteoblast lineage. We observed significantly normalized cell number in Capn4 and p27Kip1-double knockdown cells compared with in Capn4-knockdown cells (Fig. 1f). These results were consistent with our previous report in cells of the chondrocyte lineage . Collectively, knockdown of Capn4 in cells of the osteoblast lineage increases p27Kip1 protein levels, at least partially, by reducing calpain-mediated removal of p27Kip1. Moreover, reduction of p27Kip1 protein levels can, at least partially, rescue an impaired cell proliferative function of Capn4-kdn osteoblasts.
Bertoli et al showed that p27Kip1 protein levels are regulated by calpain-mediated modification of Akt/ FoxO3A phosphorylation. Ablation of Capn4 reduces phosphorylation of Akt and FoxO3A, and hypophosphorylated FoxO3A translocates from cytoplasm to nucleus, where it presumably increases p27Kip1 transcription through the direct binding of FoxO3A to the promoter of p27Kip1 and, consequently, p27Kip1 protein levels in embryonic fibroblasts . Therefore, we next examined whether knockdown of Capn4 alters p27Kip1 mRNA levels and modifies phosphorylation of Akt and FoxO3A in cells of the osteoblast lineage. Our data showed that knockdown of Capn4 increases p27Kip1 mRNA expression (Fig. 2a) and more rapidly reduces phosphorylation of Akt and FoxO3A than controls upon serum starvation (Fig. 2b) in proliferating osteoblasts prior to reaching confluence. We also semi-quantified the intensity of the protein bands corresponding to p-Akt (S473) and p-FoxO3A (T32) that was then normalized by t-Akt and t-FoxO3A protein levels, respectively, at various time points (Fig. 2c). The average rate constant for the dephosphorylation of p-Akt (S473) was 0.092±0.018 in control cells vs. 0.171±0.007 in Capn4-kdn cells; that for p-FoxO3A (T32) was 0.044±0.009 in control cells vs. 0.121±0.008 in Capn4-kdn cells. Thus, these results suggest that p-Akt and p-FoxO3A were likely dephosphorylated upon serum starvation approximately 2- and 3-times faster, respectively, in Capn4-kdn osteoblasts than controls. Collectively, p27Kip1 protein levels are also regulated by calpain-mediated modification of Akt/ FoxO3A signaling and a consequent increase of p27Kip1 mRNA levels in cells of the osteoblast lineage.
We next examined how deletion of Capn4 modifies phosphorylation of Akt. PP2A is a heterotrimer consisting of a scaffolding subunit, a variable regulatory subunit, and a catalytic subunit . PP2A regulatory subunits B56 α and γ are calpain substrates, and ablation of Capn4 increases the stability of PP2A, which thereby increases dephosphorylation of Akt in embryonic fibroblasts . Our co-immune precipitation assay showed an increased intensity of PP2A protein band (Fig. 3a) and significantly increased PP2A activity (Fig. 3b) of the protein complex precipitated with anti-Akt antibody. Collectively, these results suggest that calpain modifies p27Kip1 mRNA expression, at least in part, by modifying phosphorylation of Akt/FoxO3A in cells of the osteoblast lineage.
Calpains are cysteine proteases, which have numerous diverse substrates and regulate various cellular functions . We previously showed that osteoblast-specific Capn4 knockout mice exhibited significantly reduced trabecular and cortical bone at least partially due to reduced cell proliferation of the osteoblast lineage in vivo . However, the limitation of our report was that we failed to identify a specific molecular target of calpain responsible for their bone phenotype.
p27Kip1 has been shown to be one of those critical target molecules of calpain and that calpain activity uniquely modulates levels of p27Kip1 in vitro [11, 13, 15, 16, 18]. Consistent with our previous report in cells of the chondrocyte lineage , we here showed that ablation of Capn4 reduces cell growth by decreased cell proliferation of the osteoblast lineage. Knockdown of Capn4 increases p27Kip1 protein levels by reducing removal of p-S10-p27Kip1, a target of calpain-mediated degradation of p27Kip1, resulting in reduced phosphorylation of Rb. Additional ablation of p27Kip1 protein, at least partially, rescues the impaired cell growth phenotype of the Capn4-kdn osteoblastic cells. It was also shown previously that regulatory subunits of PP2A are calpain substrates; therefore, knockdown of calpain stabilizes PP2A protein in a protein complex assembled with Akt, which thereby promotes dephosphorylation of Akt and FoxO3A in embryonic fibroblasts . The promoter region of p27Kip1 contains binding sequences for FoxO3A . Therefore, we next examined whether calpain regulates p27Kip1 protein levels by increasing p27Kip1 mRNA levels in cells of the osteoblast lineage. Knockdown of calpain increases the intensity and activity of PP2A in a PP2A-Akt protein complex, resulting in more rapid dephosphorylation of Akt and then FoxO3A in osteoblasts upon serum-starvation. Collectively, calpain is also a critical modulator for p27Kip1 in cells of the osteoblast lineage. Figure 4 shows a hypothetical scheme of calpain-mediated regulation of p27Kip1 in cells of the osteoblast lineage. Knockdown of calpain increases PP2A protein stability and activity assembled with Akt, resulting in reducing phosphorylation of Akt and then FoxO3A. Underphosphorylated FoxO3A translocates to nucleus and increases p27Kip1 mRNA levels through direct binding to the p27Kip1 promoter and increases p27Kip1 protein levels (left). On the other hand, knockdown of calpain decreases calpain-mediated degradation of p27Kip1 (right). Increased p27Kip1 inactivates mainly cyclin D-cdk4, resulting in reduced phosphorylation of Rb and impaired cell cycle transition at G1/S.
The mechanisms by which intermittent administration of PTH shows an anabolic effect on bone have been intensively studied. Investigators reached various conclusions depending on cell lines, dose and frequency of PTH administration, cell density, and culture condition in vitro studies [19–25]. However, a line of evidence has suggested that upregulation of p27Kip1 and cyclin-dependent kinase inhibitor 1A (p21Cip1) proteins [26, 27] through PTH-mediated increase of protein kinase A (PKA) signaling is positively associated with an anabolic action of PTH in bone . Because we have showed previously that ablation of calpain significantly decreases PTH-mediated PKA signaling [9, 10], calpain may uniquely increase p27Kip1 protein levels, at least partially, through a mechanism independent of a PTH-PKA signaling pathway. Further studies will be necessary to demonstrate how calpain-mediated modulation of p27Kip1 in bone affects anabolic action of PTH in vivo; however, our results at least suggest that calpain inhibition can be a critical therapeutic target for osteoporosis because it could increase an efficiency of anabolic action of PTH by uniquely increasing p27Kip1 protein levels in cells of the osteoblast lineage.
In conclusion, our current report suggests that calpain regulates p27Kip1 protein levels by modifying p27Kip1 protein degradation and p27Kip1 transcription via phosphorylation of Akt/FoxO3A, in cells of the osteoblast lineage.
We thank Dr. Hanno Hock and Laura B Prickett-Rice at Center for Regenerative Medicine in MGH for consultation and assistance for flow cytometric analysis. This work was partially supported by National Institutes of Health Grant R01 DK072102, William F. Milton Fund, and MGH interim support fund (to M.S.).