The CXCR4 antagonist AMD3100 suppresses hypoxia-mediated growth hormone production in GH3 rat pituitary adenoma cells

Pituitary adenomas produce the chemokine stromal cell-derived factor (SDF-1 alpha/CXCL12) and its receptor, CXCR4. A recent study indicated that CXCL12 and CXCR4 are concomitantly up-regulated in hypoxia. The objective of this study was to analyze the molecular mechanism of hypoxia-mediated CXCR4 up-regulation and assess the effect of pharmacological inhibition of CXCR4 by the receptor blocker, AMD3100, on pituitary function. CXCR4 expression in pituitary adenoma tissues was determined by a tissue microarray analysis of 62 pituitary adenoma samples. CXCR4 expression was significantly elevated and positively correlated with Knosp grade in pituitary adenomas (P < 0.005), and was higher in macroadenoma and growth hormone (GH)-producing adenomas. Pre-operative serum GH levels were significantly correlated with CXCR4 levels in the microarray (P < 0.0001). The relative expression of genes/gene categories that were modulated by up-regulated CXCL12/CXCR4 signaling was determined by a comparative transcriptome analysis of wild-type and CXCR4-knockdown cells in normoxia and hypoxia using the rat GH-producing and prolactin-producing pituitary adenoma cell line, GH3. Real-time reverse transcriptase-polymerase chain reaction analysis (RT-PCR) showed that CXCR4 mRNA expression in GH3 cells was increased by hypoxia (1% oxygen), and a cDNA microarray analysis revealed that inhibin beta-C expression was diminished. siRNA-mediated CXCR4 knockdown blocked the hypoxia-induced decrease in inhibin beta-C mRNA expression, as did inhibition of CXCR4 activity with AMD3100. An ELISA study demonstrated that GH secretion by wild-type GH3 cells was moderately enhanced by hypoxia and further potentiated by exposure to recombinant SDF-1 alpha/CXCL12 protein. Conversely, hypoxia-induced GH secretion was reduced in CXCR4-silenced cells and in cells treated with the CXCR4 antagonist, AMD3100, notwithstanding the presence of SDF-1 alpha/CXCL12 protein. These latter observations reflect the failure of hypoxia to suppress expression of inhibin beta-C in cells deficient in CXCR4 or in which CXCR4 signaling was blocked. Together, these results indicate that the SDF-1 alpha/CXCL12-CXCR4 signaling pathway interfaces with the classical endocrine pathway to up-regulate GH production via suppression of inhibin beta-C. Because it blocks CXCR4 and prevents hypoxia-induced down-regulation of inhibin beta-C expression, AMD3100 has promise as a molecular-targeting agent in the treatment of GH-producing adenomas.