Endocrine Abstracts

A key component of androgen synthesis is the availability of the pro-hormone DHEA, which is either converted to active androgens or inactivated to its sulfate ester DHEAS by DHEA sulfotransferase (SULT2A1). The latter reaction requires provision of the universal sulfate donor 3′-phosphoadenosine-5′-phosphosulfate, PAPS. In humans, PAPS is generated by the PAPS synthase isoforms PAPSS1 and PAPSS2. Recently, inactivating PAPSS2 mutations have been identified in a patient with androgen excess but undetectable DHEAS¹, implicating DHEA sulfation as a gatekeeper to human androgen synthesis. To examine why ubiquitously expressed PAPSS1 cannot compensate for mutant PAPSS2, we have investigated the differential impact of PAPSS1/2 on DHEA sulfation. Firstly, we determined the subcellular localisation of PAPSS1/2 by indirect immunofluorescence in NCIh295R, HepG2, and HEK293 cells, derived from adrenal, liver and kidney, respectively. We found that PAPSS1 invariably showed nuclear localisation whereas PAPSS2 was predominantly cytosolic in HEK293 cells. By contrast, in adrenal and liver cells, capable of DHEA sulfation by cytosolic SULT2A1, PAPSS2 was almost exclusively nuclear. We generated bicistronic constructs for concurrent overexpression of SULT2A1 with PAPSS1/2 in HEK293 cells that lack SULT2A1 or PAPSS2 and only have low endogenous PAPSS1. Realtime PCR demonstrated successful overexpression of all targets (ΔCts 6–8). Enzymatic activity assays measuring conversion of tritiated DHEA to DHEAS demonstrated that SULT2A1 overexpression provides HEK293 cells with some ability to sulfate DHEA. However, concurrent overexpression of SULT2A1 with PAPSS yielded a twofold (PAPSS1) and fivefold (PAPSS2) increase in DHEA sulfation rate. Differential siRNA knockdown of both PAPSS isoforms in the adrenal NCIh295 TR cell line demonstrated upregulation of PAPSS1 mRNA by PAPSS2 knockdown and vice versa. These findings demonstrate a tight co-regulation of PAPSS isoforms in support of sulfation and suggest that a nuclear localisation of PAPSS2 is a precondition for efficient DHEA sulfation.

Reference

1. NEJM 2009 360 22 2310–8.