Suberoylanilide hydroxamic acid (SAHA; vorinostat) causes bone loss by inhibiting immature osteoblasts

Abstract

Histone deacetylase (Hdac) inhibitors are used clinically to treat cancer and epilepsy. Although Hdac inhibition accelerates osteoblast maturation and suppresses osteoclast maturation in vitro, the effects of Hdac inhibitors on the skeleton are not understood. The purpose of this study was to determine how the pan-Hdac inhibitor, suberoylanilide hydroxamic acid (SAHA; a.k.a. vorinostat or Zolinza™) affects bone mass and remodeling in vivo. Male C57BL/6 mice received daily SAHA (100 mg/kg) or vehicle injections for three to four weeks. SAHA decreased trabecular bone volume fraction and trabecular number in the distal femur. Cortical bone at the femoral midshaft was not affected. SAHA reduced serum levels of P1NP, a bone formation marker, and also suppressed tibial mRNA levels of type I collagen, osteocalcin and osteopontin, but did not alter Runx2 or osterix transcripts. SAHA decreased histological measures of osteoblast number but interestingly increased indices of osteoblast activity including mineral apposition rate and bone formation rate. Neither serum (TRAcP 5b) nor histological markers of bone resorption were affected by SAHA. P1NP levels returned to baseline in animals which were allowed to recover for four weeks after four weeks of daily SAHA injections, but bone density remained low. In vitro, SAHA suppressed osteogenic colony formation, decreased osteoblastic gene expression, induced cell cycle arrest, and caused DNA damage in bone marrow-derived adherent cells. Collectively, these data demonstrate that bone loss following treatment with SAHA is primarily due to a reduction in osteoblast number. Moreover, these decreases in osteoblast number can be attributed to the deleterious effects of SAHA on immature osteoblasts, even while mature osteoblasts are resistant to the harmful effects and demonstrate increased activity in vivo, indicating that the response of osteoblasts to SAHA is dependent upon their differentiation state. These studies suggest that clinical use of SAHA and other Hdac inhibitors to treat cancer, epilepsy or other conditions may potentially compromise skeletal structure and function.

Runx2 Protein Represses Axin2 Expression in Osteoblasts and Is Required for Craniosynostosis in Axin2-deficient Mice*

“…Expression levels of mRNAs for Runx2, Axin2, and Gapdh were measured by real-time PCR. Primer sequences were reported previously (32,33). Reactions were performed using 37.5 ng of cDNA/15 l with Bio-Rad iQ SYBR Green Supermix and the Bio-Rad MyiQ single color real-time PCR detection system.…”

“…For gene expression studies, RNA was harvested with TRIzol reagent (Invitrogen) after 7, 14, or 21 days in osteogenic culture. RNA was reverse transcribed and expression levels of Runx2 or Bglap (genes associated with osteoblast maturation) were quantified as described above and previously (32).…”

Histone deacetylases (HDAC) in physiological and pathological bone remodelling

“…Using an in vivo model, it was demonstrated that vorinostat resulted in bone loss and reduced osteoblast numbers in mice [77]. Despite a negative effect on the immature osteoblasts, an increase in local bone formation was observed, with mature osteoblasts possibly being resistant to HDACi-induced apoptosis [77]. Selective targeting and lower doses (10-100x lower) of HDACi have been shown to be better tolerated in animal models and have different effects on bone cells without the induction of apoptosis.…”

Histone Deacetylase Inhibitors Target the Leukemic Microenvironment by Enhancing a Nherf1-Protein Phosphatase 1α-TAZ Signaling Pathway in Osteoblasts

“…HDACi are known to directly target cancer cells by increasing expression of many proteins including those that regulate tumor suppression, DNA repair, and cell-cycle arrest. Because HDACi have also been shown to alter expression of many genes within cells of the osteoblast lineage (15)(16)(17)(18), we hypothesized that HDACi might also target the leukemic microenvironment. HDACi have been previously shown to up-regulate Nherf1 (also known as EBP-50), a scaffold protein mutated in human hypophosphatemic nephrolithiasis/osteoporosis type 2.…”