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, 156 (3), 1033-9

PK11195 Effect on Steroidogenesis Is Not Mediated Through the Translocator Protein (TSPO)

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PK11195 Effect on Steroidogenesis Is Not Mediated Through the Translocator Protein (TSPO)

Lan N Tu et al. Endocrinology.

Abstract

Translocator protein (TSPO) is a mitochondrial outer membrane protein of unknown function with high physiological expression in steroidogenic cells. Using TSPO gene-deleted mice, we recently demonstrated that TSPO function is not essential for steroidogenesis. The first link between TSPO and steroidogenesis was established in studies showing modest increases in progesterone production by adrenocortical and Leydig tumor cell lines after treatment with PK11195. To reconcile discrepancies between physiological and pharmacological interpretations of TSPO function, we generated TSPO-knockout MA-10 mouse Leydig tumor cells (MA-10:TspoΔ/Δ) and examined their steroidogenic potential after exposure to either dibutyryl-cAMP or PK11195. Progesterone production in MA-10:TspoΔ/Δ after dibutyryl-cAMP was not different from control MA-10:Tspo+/+ cells, confirming that TSPO function is not essential for steroidogenesis. Interestingly, when treated with increasing concentrations of PK11195, both control MA-10:Tspo+/+ cells and MA-10:TspoΔ/Δ cells responded in a similar dose-dependent manner showing increases in progesterone production. These results show that the pharmacological effect of PK11195 on steroidogenesis is not mediated through TSPO.

Figures

Figure 1.
Figure 1.
CRISPR/Cas9-mediated deletion of TSPO in steroidogenic MA-10 Leydig cells. A, Sequence of Tspo exon 2 showing the start codon and targeted sequence followed by the protospacer adjacent motif. B, After transfection with Tspo-CRISPR/Cas9 construct, MA-10 clones were screened for TSPO protein expression using a monoclonal TSPO antibody that recognizes amino acids 156–169 encoded by Tspo exon 4. Western blots that identified the three TSPO-deleted clones are shown. Control bands are β-Actin (ACTB). C, Sequencing results of Tspo cDNA in 3 MA-10:TspoΔ/Δ clones confirmed indel mutations in the target sites of expressed alleles. Regions corresponding to guide RNA sequences are shown in green, deletions and insertions are shown in red. Deleted regions ranged from 2 bp in MA-10:TspoΔ/Δ1 to 88 bp in MA-10:TspoΔ/Δ3. D, Mutations in Tspo induced a frame shift and/or generated a stop codon, which resulted in a truncation with fragments restricted to TSPO N-terminal 28 and 25 amino acids for alleles in MA-10:TspoΔ/Δ1, 21 and 28 amino acids for alleles in MA-10:TspoΔ/Δ2, and 23 amino acids for the one allele in MA-10:TspoΔ/Δ3. The second allele in MA-10:TspoΔ/Δ3 was a Gly28 mutation that induced a significant change to the short α helix loop (arrowhead) that connects transmembrane region 1 and 2 that ultimately led to complete loss of TSPO protein expression. E, Representative light microscopy images of MA-10:Tspo+/+ and MA-10:TspoΔ/Δ cells showed that MA-10:TspoΔ/Δ cells were healthy with no apparent morphological changes (Scale bar: 200 μm). F, Immunocytochemistry confirmed complete absence of TSPO in the different MA-10:TspoΔ/Δ clones (Scale bar: 20 μm).
Figure 2.
Figure 2.
TSPO deficiency does not affect progesterone production in MA-10 cells. A, TSPO deletion in the three MA-10:TspoΔ/Δ clones did not affect baseline or Bt2cAMP-stimulated progesterone production. Bt2cAMP stimulation increased progesterone levels to a similar extent in both intact MA-10:Tspo+/+, and TSPO-deleted clones: MA-10:TspoΔ/Δ1, MA-10:TspoΔ/Δ2, and MA-10:TspoΔ/Δ3. Progesterone levels after Bt2cAMP treatment in clone MA-10:TspoΔ/Δ3 were significantly higher than MA-10:Tspo+/+ cells. Mean values were generated from two to three experiments conducted in triplicate. B, Western blots showing absence of TSPO and the induction of STAR after Bt2cAMP stimulation of MA-10:TspoΔ/Δ clones and MA-10:Tspo+/+ cells. Induction of STAR expression after Bt2cAMP stimulation were identical in the different MA-10:TspoΔ/Δ clones compared with MA-10:Tspo+/+ cells. Control bands are β-Actin (ACTB).
Figure 3.
Figure 3.
PK11195 induced steroidogenesis in the absence of TSPO. PK11195 dose-dependent increase in progesterone production was observed in both MA-10:TspoΔ/Δ clones (1, 2, and 3) and control MA-10:Tspo+/+ cells. Mean values were generated from three to four independent experiments conducted in triplicate.

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