Network Analysis of Dedifferentiation of Parotid Acinar Cells

Salivary gland function is disrupted in Sjogren's Syndrome and head/neck cancer radiation patients, creating a need to understand how to re-differentiate or replace salivary tissue. Salivary acinar cells lose their tissue specific function in culture, however, the molecular basis for this dedifferentiation is unknown. OBJECTIVES: To identify genetic networks associated with the loss of acinar cell function during culture. METHODS: Rat parotid glands were digested with collagenase and hyaluronidase and primary cells cultured on collagen-coated plates. To define culture conditions, cells were grown in growth factor and hormone-supplemented media. RNA was isolated for RT-PCR analysis of Mist1, amylase and Parotid Secretory Protein (PSP) expression. To determine global gene expression patterns, primary cultures were collected at 0h, 6h and 18h in each of 3 independent experiments, and RNA isolated for Affymetrix microarray analysis. Partek was used for normalization and statistical analysis of the microarray data, and Ingenuity Pathway Analysis used to create networks based on a knowledge base of published interactions. RESULTS: Expression of markers of differentiation (Mist1, amylase, and PSP) significantly (P<0.05) decreased in all culture media from 0h to 24h suggesting that loss of differentiation is independent of culture conditions. Network analysis comparing 0h and 6h microarray results indicate a strong decrease in genes involved with cell proliferation. However, comparison of the 6h and 18h microarray results yields a different network indicating downregulation of several transcription factors, including FOXO1A, ELF3, and KLF10, suggesting that they may be involved in differentiation of acinar cells. Decreased expression of transcription factors, ELF3, KLF10, Mist1 and FOXO1A was confirmed by RT-PCR. CONCLUSIONS: Dedifferentiation of primary parotid cells occurs rapidly in all culture media tested. Gene expression microarray based networks identify three novel candidate transcription factors (FOXO1A, ELF3, and KLF10) as being associated with differentiation of acinar cells. Support: NIDCR DE012205.