Figure 3. Sclerostin is rapidly degraded by the lysosome following bone anabolic stimuli.

(A) Ocy454 cells transfected with GFP-sclerostin were treated with cycloheximide (150 µg/mL) to prevent new protein synthesis and either DMSO (0.1%), bafilomycin A1 (100 nM) to inhibit lysosomal degradation, brefeldin A (2 μm) to inhibit secretion, or MG-132 (10 μm) to inhibit the proteasome 4 hr prior to FSS. Cells were subjected to 5 min of FSS at 4 dynes/cm² and lysed immediately after the end of FSS or 30 min after the conclusion of FSS. Western blots were probed for sclerostin and β-actin. Time courses show mean ± SEM (n = 3–6 independent experiments/group). (B) Ocy454 cells transfected with GFP-sclerostin were pre-treated with DMSO (0.1%) or bafilomycin A1 (100 nM) to inhibit lysosomal degradation for 30 min prior to the addition of vehicle or PTH (1–34) (10 nM) for an additional 30 min (n = 3). Sclerostin abundance relative to the loading control was quantified. Graph depicts mean ± SD. *p<0.05, **p<0.01, ****p<0.0001 by two-way ANOVA with Holm–Sidak post hoc correction. (C) Amino acid sequences for sclerostin from mouse, human, rat, cow, and chicken were aligned using NCBI COBALT. Abbreviated sequences are shown and are annotated for putative lysosomal signal sequences. Full sequences are presented in Figure 3—figure supplement 1.

Figure 3—figure supplement 1. Sclerostin is rapidly degraded by the lysosome.

(A) UMR106 cells were treated with cycloheximide (150 µg/mL) and either DMSO (0.1%), bafilomycin A1 (100 nM) for 6 hr, or leupeptin (200 μM) for 4 hr prior to FSS. Cells were subjected to 1 min of FSS at 4 dynes/cm² and were lysed 5 or 10 min after the conclusion of FSS. Western blots were probed for sclerostin. Sclerostin abundance relative to total protein was quantified. For each antibody, blots are from a single gel and exposure; a vertical black line indicates removal of irrelevant lanes. (B) UMR106 cells were treated with cycloheximide (150 µg/mL), and lysates were collected at 0, 1, 2, and 4 hr after treatment in the absence of stimuli. Whole-cell lysates were western blotted for sclerostin abundance (n = 2). Sclerostin abundance relative to the total protein was quantified. Graph shows mean ± SD and best-fit linear regression constrained through y = 1. (C) Full amino acid sequences for sclerostin from mouse, human, rat, cow, and chicken were aligned using NCBI COBALT. Putative lysosomal signal sequences are annotated. The secretory signal peptide is annotated for amino acids 1–23.