Molecular, Biophysical and Regulatory Characterisation of Ion Transport Pathways in the Human Immortalised Apocrine Gland Secretory Cell Line, ASG5

In humans, sweat is produced by two different sweat glands: the eccrine glands which help cool the body by secreting a hypotonic salty fluid directly onto the skin surface (thermoregulatory sweating), and the apocrine glands whose function is less clear. Apocrine glands located in the axillae secrete an odourless, milky fluid into the lumen of the hair follicle, and this is subsequently metabolised by the axillary microbiota to produce malodorous compounds. Apocrine glands do not participate in thermoregulation, and little is known about the molecular mechanisms driving secretion. However, it is primarily driven by the loss of lipid-containing vesicles from the lumenal pole of each cell, a process referred to as 'apocrine' secretion. There is also a fluidic component which flushes vesicular material toward the glandular duct.

The paucity of data concerning apocrine secretory pathways correlates with the difficulty in obtaining tissue to study. In recent years, Unilever have developed a unique apocrine cell line (the ASG5 cell), which is a robust in vitro model, representative of native tissue (1).

The objective of the current project is to use ASG5 cells to identify and characterise the ion transport proteins contributing to the production of the fluidic component of apocrine gland secretion, and to determine how known secretory agonists regulate them. The ASG5 cell line has supported two PhD projects to date. These objectives will be achieved by using molecular techniques (qPCR, Western blotting and immunohistochemistry) to identify the electroneutral and charge-carrying plasmalemmal ion transport proteins present in ASG5 cells, and fluorescent imaging of intracellular pH (pHi) and patch clamp techniques (whole cell or cell-attached configurations) to assess transporter and channel regulation and inhibitor sensitivity. SiRNA knockdown will be used to delineate the relative contributions of different isoforms of the same transport protein, and phosphoproteome screening to determine the cellular effects of different secretagogues. Novel data generated from these approaches will feed into molecular modelling and database screening at Unilever Port Sunlight which will identify cosmetic analogues of known transporter inhibitors and potential technology options for modulating malodour and health conditions such as bromhidrosis.

The broad objectives and work plan are as follows:
Milestone 1. Development of cell culture techniques and molecular identification of ion channel and electroneutral ion transport proteins. Develop cell culture techniques for the apocrine secretory cell line ASG5. Use PCR, Western and IHC to identify the isoforms and location of the ion channel and electroneutral transport proteins present (eg. Na/K-ATPases, NHEs, NKCC1, AE2, Best2 / ANO1 channels, K+, Cl, Ca channels).

Milestone 2. Characterisation of ion channel / transporter activity by pHi and patch clamp measurements. Utilise pHi fluorescence measurement to determine the activity and inhibitor sensitivity of selected electroneutral transporters. Use siRNA knockdown to determine the contribution of different isoforms. Develop and use patch clamp techniques to characterise Cl- and K+ channels using the same approach.

Milestone 3. Characterisation of ion channel / transporter regulation by known apocrine secretory agonists. Use pHi and patch clamp techniques to determine quantitatively the effect of known apocrine secretory agonists (eg. adrenaline, estradiol, testosterone) on ion channel and transporter activity. Determine the effects of selected agonists on the phosphoproteome of stimulated ASG5 cells.

Milestone 4. Identification of cosmetic equivalent ion transporter inhibitory compounds by molecular modelling. Screen cosmetic active databases for equivalents of known pharmaceutical inhibitors of selected target proteins identified in ASG5 cells (Unilever Port Sunlight).

1. Evans, R.L et al (2007) Patent GB200705738 A