Integrin receptor ligand nephronectin plays a role in aldosterone production in hypertension and protects adrenal carcinoma cells against death

Objective: 5%-10% of hypertension is due to adrenal aldosterone-producing adenomas (APAs), which are potentially curable by unilateral adrenalectomy. Recent discoveries of somatic mutations permit recognition of sub-types of APAs with different characteristics and underlying pathology [1]. In comparing classical zona fasciculata (ZF)-like APAs with the histologically- and biochemically- different zona glomerulosa (ZG)-like APAs, we hypothesized that specific genes would be found responsible for elevating aldosterone production in the latter, more recently recognised, APAs, which secrete more aldosterone per unit volume than classical APAs.

Methods: A microarray was performed comparing five ZG-like APAs with eight ZF-like APAs, as well as a second microarray comparison of normal adrenal ZG and ZF cells to identify genes involved in aldosterone production. Findings were validated by quantitative PCR (qPCR) and immunohistochemistry. The role of these genes on aldosterone production was assessed by transfecting human adrenocortical carcinoma cell-line H295R, with differences in aldosterone production measured by homogeneous time-resolved fluorescence. Cell viability was measured by the CASY Cell-Counter and IncuCyte Live Cell Imaging, while cell death was assessed by Annexin V-Propidium Iodide dual-staining.

Results: Microarray analyses identified the top gene as Nephronectin (NPNT) (x12.2, p= 2.97E-08) - a secreted extracellular matrix protein known to be a ligand of integrin alpha8-beta1 receptor [2]. qPCR confirmed that NPNT was 25.3-fold more highly expressed in normal ZG vs ZF (P=0.0001) and 29.9-fold upregulated in the APAs producing more aldosterone (P=0.00034). At protein level, NPNT staining outlined groups of cells, or glomerular ‘rotundules’ in all cases. RNA analyses and immunohistochemistry of human adrenals also showed NPNT expression to be almost entirely switched off in the ZG adjacent to an APA, possibly as a form of negative feedback. Functionally, over expression of NPNT in H295R cells led to approximately doubling of aldosterone (P=0.03) and conversely, silencing resulted in 20% reduction in hormone production (P=0.0004). Silencing NPNT also resulted in a significant reduction in cellular adhesion in the early stages and approximately 40% increase in cell death after 24h. Silencing of its receptor, integrin alpha8-beta1, also led to a significant reduction in aldosterone production. Additionally, NPNT was also found to be associated with Wnt signalling, with nearly four-fold increase in APAs with a Wnt-activating mutation (P=0.0061).

Conclusion: Phenotypically and genotypically-distinct from ZF-like APAs, small NPNT-rich APAs have a greater capacity for aldosterone production. NPNT’s striking peri-glomerular distribution and ZG-selectivity point to a role in facilitating cell clustering into functional units for aldosterone secretion. In addition, NPNT plays a pivotal role in promoting cellular adhesion, probably protecting against cell death. These diverse roles of NPNT could be via the integrin or Wnt signalling pathway, or an interaction between the two. The discovery of NPNT in the adrenal was of initial value as a marker confirming the two subtypes of tumours. But excitingly NPNT has proved to be not just a marker, but critical to regulation of adrenocortical cell function and turnover. By elucidating its role, we are uncovering the pathogenesis of adrenal tumours, and may be highlighting a potential therapeutic target, either the ligand or its receptor, for adrenal cancer and hypertension.

(1) Azizan EA et al. Nature Genetics 2013; 45(9): 1055-1060.

(2) Brandenberger R et al. J. Cell Biol. 2001; 154: 447-458.