Midazolam inhibits chondrogenesis via peripheral benzodiazepine receptor in human mesenchymal stem cells

Abstract Midazolam, a benzodiazepine derivative, is widely used for sedation and surgery. However, previous studies have demonstrated that Midazolam is associated with increased risks of congenital malformations, such as dwarfism, when used during early pregnancy. Recent studies have also demonstrated that Midazolam suppresses osteogenesis of mesenchymal stem cells (MSCs). Given that hypertrophic chondrocytes can differentiate into osteoblast and osteocytes and contribute to endochondral bone formation, the effect of Midazolam on chondrogenesis remains unclear. In this study, we applied a human MSC line, the KP cell, to serve as an in vitro model to study the effect of Midazolam on chondrogenesis. We first successfully established an in vitro chondrogenic model in a micromass culture or a 2D high‐density culture performed with TGF‐β‐driven chondrogenic induction medium. Treatment of the Midazolam dose‐dependently inhibited chondrogenesis, examined using Alcian blue‐stained glycosaminoglycans and the expression of chondrogenic markers, such as SOX9 and type II collagen. Inhibition of Midazolam by peripheral benzodiazepine receptor (PBR) antagonist PK11195 or small interfering RNA rescued the inhibitory effects of Midazolam on chondrogenesis. In addition, Midazolam suppressed transforming growth factor‐β‐induced Smad3 phosphorylation, and this inhibitory effect could be rescued using PBR antagonist PK11195. This study provides a possible explanation for Midazolam‐induced congenital malformations of the musculoskeletal system through PBR.

properties. 4,5 They produce a sedation effect primarily through modulation of gamma-aminobutyric acid (GABA) receptors in the central nervous system 6,7 to block nerve impulses. GABA binds with GABA A , GABA B and GABA C receptors. Among them, the GABAA receptor is of particular significance for psychopharmacology because it contains a variety of binding sites at which behaviour-modifying drugs act to produce some or all of their effects. 8 Benzodiazepines bind to benzodiazepine sites on GABA receptors and allosterically modulate the response of the channel upon GABA binding. 9 The function of benzodiazepines acting on GABAA receptors is to increase the amplitude or decay time of GABA-mediated inhibitory post-synaptic potentials, thus increasing the inhibitory tone of GABAergic synapses to reduce the firing of neuron populations. 10 In addition to the central nervous system, benzodiazepines act on peripheral tissues performed with peripheral benzodiazepine receptor (PBR) or mitochondria translocator proteins. 11,12 A PBR is distinct in its pharmacological, anatomical, structural and physiological aspects when compared with central benzodiazepine receptors. 12,13 PBR is an 18 kD protein, localize to the mammalian mitochondria membrane, and are highly conserved among various mammalian species in various types of peripheral tissue. 11 It has been shown that PBR distributes ubiquitously in most types of tissues, including bone marrow stromal cells. 14 Putative PBR functions are involved in numerous types of physiological processes, such as steroidogenesis, apoptosis, cell proliferation, regulation of mitochondrial membrane potential, mitochondria respiratory chains, voltagedependent calcium channels and microglial activation. 11,12,[15][16][17] PK11195 is a competitive antagonist that specifically binds to PBR 18 and has been shown to reverse the inhibitory effect of Midazolam on the anxiolytic and antidepressant effects of Midazolam. 19 Although numerous papers have proved the safety of benzodiazepine and its derivatives, there remain in vitro and in vivo studies indicating the toxic effects that can occur after benzodiazepine treatment. 20,21 For example, previous study has demonstrated that the incident rate of congenital malformations was 0.23% if the maternal plasma was diagnosed as benzodiazepine positive. 21 Furthermore, Deck et al summarized the database of all infants born with major congenital malformations to mothers on antiepileptic drugs from Boston Medical Center from the years 2003 to 2010, including cleft lips/ palate, cardiac defects and urogenital defects. Results showed that for women on benzodiazepine monotherapy during pregnancy, major congenital malformations were high and extended to 10.6%. Among their infants, the rate for cleft lip/palate was approximately 0.747%. 22 Recent in vitro and in vivo animal studies have suggested that exposure to clinically relevant general anaesthetics at the peak of brain development could be detrimental to immature mammalian neurons as demonstrated by massive and widespread apoptotic neurodegeneration. 23 In addition, previous studies exposing young mice to benzodiazepine derivatives, either treated alone or together with other chemicals, have resulted in high birth rates of runts, increased retarded ossification of skull bones and a variety of sternal defects. 24,25 More importantly, Midazolam has been found to inhibit osteogenesis of human bone marrow-derived mesenchymal stem cells (hMSCs). 26 However, the detailed mechanisms for benzodiazepine-regulated bone/cartilage differentiation that associated with foetal growth retardation remain unknown.
As mentioned above, benzodiazepine and its derivatives are associated with birth defects and cleft lip/palates 27  This type of cell line has been used in studies of stem cell biology and regenerative medicine. 30 The cell line used in this study was kindly provided by Dr. Shih-Chieh Hung (China Medical University).
We also purchased primary human bone marrow-derived MSCs from Lonza Walksville Inc., which were maintained in growth medium: DMEM low glucose containing 10% foetal bovine serum (GIBCO with selected lot), 100 units/mL penicillin and 100 lg/mL streptomycin (both from GIBCO) at 37°C with a humidified 5% CO 2 atmosphere and medium was changed twice a week. Only early passages (passages 3-6) of primary hMSCs were used in this experiment. To induce chondrogenic differentiation, KP cells or hMSCs were treated with chondrogenic induction medium (DMEM low glucose, 0.1 lmol/L Dexamethasone, 50 lmol/L L-ascorbic acid-2phosphate, 19 Insulin-Transferrin-Selenium, 40 lg/mL L-proline, 1009 L-glutamine, 5 ng/mL transforming growth factor-b3). 31,32 For the pelleted culture, 3 9 10 5 cells per pellet were placed in a 15-ml centrifuge tube and centrifuged to produce a pellet. The medium was changed every 3 days. For high-density cultures, cells were seeded at 3 9 10 4 cells/cm 2 overnight and the medium was replaced with chondrogenic induction medium the following day. 31 The chondrogenic differentiation was accessed after 7 or 14 days of treatment. 31,32  guanidine hydrochloride overnight at 4°C. The absorbance of dissolved stained GAG was then quantified using a spectrophotometer at OD 620 nm. 33

| Western blot analysis
After treatment, cells were rinsed twice using chilled PBS and then lysed and collected using a radioimmunoprecipitation assay buffer (Thermo) plus protease and phosphatase inhibitor cocktails (Thermo).
After sonication, cell lysate was centrifuged at 15 000 9 g for 30 minutes at 4°C and supernatant was collected in an eppendorf tube and stored at À80°C. Protein concentration was assessed using a bicinchoninic acid protein assay (Bio-Rad) as per the manufacturer's instructions. Fifteen to thirty lg of protein was resolved using SDS-PAGE, followed by electro-transferred onto a methanol-soaked polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% milk or 2% bovine serum albumin (for phosphorylated protein) in Tris-Buffered Saline-Tween 20 (TBST) and then incubated with primary antibody overnight at 4°C. The membrane was then rinsed with TBST and the immunocomplex on membrane was detected using horse-radish peroxidase-conjugated secondary antibody, and the final immunocomplexes were visualized using fluorography with an enhanced chemiluminescence reagent (GE Healthcare Life Sciences).

| Immunostaining and immunofluorescence
Cells in micropellet cultures were fixed with 4% paraformaldehyde, washed in PBS and then sectioned and frozen (LEICA CM 1950).
The 10-lm-thick sections were rinsed twice in PBS, followed by soaking in SuperBlock blocking buffer (Thermo) for 1 hour at room temperature, and then incubated with primary antibody overnight at 4°C. The sections were then rinsed twice with PBS and stained with secondary antibody conjugated with Alexa 594 (Thermo). Images were taken using an Olympus epifluorescence microscope. For highdensity cultures, cells were rinsed twice with PBS and then fixed with 4% paraformaldehyde, soaked in SuperBlock blocking buffer (Thermo) for 1 hour at room temperature and then incubated with primary antibodies overnight at 4°C. The sections were then rinsed twice with PBS and stained with secondary antibody conjugated Alexa 594 (Thermo). Images were taken using an Olympus epifluorescence microscope or confocal microscope (Olympus FV-1000).

| Statistics
All quantified results are shown in mean AE SEM of three to four independent experiments. Statistical analyses were performed using ANOVA followed by Tukey's test for significant difference. Significance was accepted when P < .05.   Figure 3A,B). In addition, chondrogenic inductioninduced type II collagen was dose-dependently inhibited by the treatment of Midazolam ( Figure 3C). Similar was confirmed using micropellet culture of primary hMSCs ( Figure S2). Together, these results demonstrate that Midazolam-inhibited chondrogenic differentiation is reproducible in MSCs from different sources.

| Establishment of chondrogenic differentiation
Because the cell type that we used was taken from peripheral tissue, we sought to understand whether Midazolam-inhibited  36 We found that administration of Midazolam significantly suppressed TGF-b-induced Smad3 phosphorylation dose-dependently ( Figure 6A). Interestingly, if cells were co-treated with PBR antagonist PK11195, the Midazolam-induced suppression of Smad3 phosphorylation could be rescued ( Figure 6B   Although benzodiazepine derivatives may cause musculoskeletal defects, the molecular mechanisms of how these benzodiazepine derivatives result in congenital defects remain unknown. To confirm the inhibitory effect of Midazolam, we applied TGF-b-containing chondrogenic induction medium 31,32 in human MSCs. We used several differentiation markers to indicate chondrogenesis, including SOX9, type II collagen and an increased amount of GAGs, which could be stained using Alcian blue. SOX9 is a transcriptional factor that plays a key role in the regulation of chondrogenesis, and multiple signalling pathways are known to regulate the expression and activity of SOX9 during chondrogenesis [39][40][41][42] . Activated SOX9 in turn regulates chondrogenic-dependent markers, such as type II collagen 43,44 and aggrecan. 45 In addition, SOX9, together with SOX5 and SOX6 to form SOX trios, and expression of these trios lead to chondrogenesis. 34,35 In current study, we found that Midazolam inhibits protein levels of SOX9 We also investigated how Midazolam acts on MSCs to suppress their chondrogenic differentiation. Because MSCs were isolated from peripheral tissues, we aimed for the PBR. Early reports showed that PBR is expressed ubiquitously in most types of tissues, including bone marrow 14 and is required for adipogenic differentiation and MSC proliferation. 49 It is also known that PBR expresses in murine osteoblasts and osteoclasts, suggesting that PBR may be involved in bone homeostasis. 50 To confirm that PBR mediated inhibitory effects The major signalling pathway used to induce chondrogenesis in this study was TGF-b signalling. TGF-b, a pleiotropic cytokine, is involved in the regulation of cell division and suppression of immune response. 55 Upon binding of TGF-b to its receptor, the receptor complex recruits and phosphorylates the carboxy terminus of receptor-regulated Smad proteins (R-Smads: Smad2 and Smad3). 36,56 Phosphorylated Smads can interact with Smad4 to become a complex, and this complex translocates into the nucleus and turns on gene transcription. 55 Smad3 is able to activate SOX9 via recruitment of CREB-binding protein/p300. 46 In addition, TGF-b/Smad signalling is essential for osteocartilage development, bone homeostasis and MSC fate decision. 57 Disruption of TGF-b/Smad signalling in vivo resulted in serious problems in the musculoskeletal system. 58 Midazolam has been found to inhibit protein phosphorylation. For example, a previous report indicated that Midazolam inhibits platelet activation by inhibiting protein phosphorylation of protein kinase C. 59 In mouse hippocampal slices, Midazolam was found to inhibit extracellular signal-related kinase 1/2 activity, and this inhibition was likely mediated via N-methyl-d-aspartate receptors, phospholipase C and PKC-dependent signalling. 60 We therefore proposed that the chondrogenic inhibitory effect of Midazolam was caused by the inhibition of Smad 3 phosphorylation. Indeed, Midazolam can dosedependently inhibit Smad3 phosphorylation ( Figure 6) and this Smad3 phosphorylation inhibition was mediated by PBR, as proved when administration of PK11195 reversed the inhibitory effect of Midazolam. We therefore speculate that the inhibitory effect of Midazolam on TGF-b-induced Smad 3 phosphorylation is mediated by binding to PBR. Several studies have demonstrated that in most types of tissue, PBR is primarily located in the outer membrane of mitochondria. 13 In addition to the outer mitochondrial membrane, F I G U R E 6 Midazolam inhibits transforming growth factor (TGF)-b-induced Smad3 phosphorylation. KP cells were seeded at 3 9 10 4 cells/cm 2 , serum starved for 16 h and then treated with TGF-b (5 ng/mL) in the presence of various doses of Midazolam (0, 1, 10 and 20 lmol/L) for 1 h. (A) Levels of Smad3 phosphorylation; total Smad3 and vinculin were analysed using Western blot analysis. Quantification results are presented as mean AE SEM of three independent experiments (*P ≦ .05, **P ≦ .01, ***P ≦ .005). (B) KP cells were seeded at 3 9 10 4 cells/cm 2 , serum starved for 16 h and then treated with TGF-b (5 ng/mL) in the presence of Midazolam (10 lmol/L, MDZ) or co-treated with PK11195 (10 lmol/L, PK). Levels of Smad3 phosphorylation; total Smad3 and vinculin were analysed using Western blot analysis. Quantification results are presented as mean AE SEM of three independent experiments (*P ≦ .05, **P ≦ .01, ***P ≦ .005) PBR was also located in various subcellular regions. Mukherjee and Das first reported that although the amount of [ 3 H]-labelled R05-4864 (another PBR ligand) binding sites was highest in the mitochondrial fraction, the nuclear and cytosolic fractions also contained significant amounts of these binding sites. 61 A study conducted by Kuhlmann and Guilarte discovered that after neural injury, PBR localization was in cytosol or perinuclear areas in microglia cells and macrophages. 62 In addition, subcellular localization of PBR was also found in cytosol or in the nuclei of different cell types (ie MA-10, MCF-7 and MDA-MB-231), and these nuclear localized PBR was implicated to correlate with cell proliferation and nuclear cholesterol transport. 63 This cytosolic localization of PBR supports our speculation that Midazolam can bind with cytosolic PBR to regulate TGF-binduced Smad3 phosphorylation, which also occurs in cytosol.
Further study of how Midazolam binds to cytosolic PBRs and exerts its function should be investigated.
Taken together, our findings suggest that Midazolam inhibits chondrogenesis via PBR to inhibit TGF-b signalling, which in turn suppresses chondrogenesis. This partially explains why this sedative drug can result in congenital malformation of newborns if a pregnant woman is treated with benzodiazepine for anxiety and insomnia.
Thus, the future application of these types of drugs must be more thoroughly considered. Further studies to elucidate how these benzodiazepine derivatives interact with their receptors and cause various outcomes require more sophisticated investigation.