Radiation therapy for head and neck cancers results in permanent damage to the saliva producing acinar compartment of the salivary gland. To date, a pure pro-acinar cell line to study underlying mechanisms of acinar cell differentiation in culture has not been described. Here, we report the establishment of a pro-acinar (mSG-PAC1) and ductal (mSG-DUC1) cell line, from the murine submandibular salivary gland (SMG), which recapitulate developmental milestones in differentiation. mSG-DUC1 cells express the ductal markers, keratin-7 and keratin-19, and form lumenized spheroids. mSG-PAC1 cells express the pro-acinar markers SOX10 and aquaporin-5. Using the mSG-PAC1 cell line, we demonstrate that FGF2 regulates specific steps during acinar cell maturation. FGF2 up-regulates aquaporin-5 and the expression of the α3 and α6 subunits of the α3β1 and α6β1 integrins that are known to promote SMG morphogenesis and differentiation. mSG-DUC1 and mSG-PAC1 cells were derived from genetically modified mice, homozygous for floxed alleles of the integrin α3 subunit. Similar to SMGs from α3-null mice, deletion of α3 alleles in mSG-PAC1 cells results in the up-regulation of E-cadherin and the down-regulation of CDC42. Our data indicate that mSG-DUC1 and mSG-PAC1 cells will serve as important tools to gain mechanistic insight into salivary gland morphogenesis and differentiation.
The salivary gland can be permanently impaired by radiation treatment for head and neck cancers. Efforts at tissue regeneration have focused on saliva-producing acinar cells. However, myoepithelial cells are also critical to gland function, but mechanisms that regulate their differentiation are poorly defined. To study myoepithelial differentiation, we employed mSG-PAC1 murine salivary gland epithelial cells. We demonstrate that mSG-PAC1 spheroids exhibit phenotypic plasticity between pro-acinar and myoepithelial cell fates. Increased expression of pro-acinar/acinar or myoepithelial RNAs was identified from spheroids cultured under different media conditions by microarray followed by gene-set enrichment analysis. Spheroids cultured with different medium components expressed proteins typical of either acinar or myoepithelial cells, as detected by immunocytochemistry. We demonstrate that the pattern of TAZ expression in the epithelial compartment of the differentiating murine salivary gland correlates with the expression of the myoepithelial marker alpha-SMA, as is the case for TAZ expression in mSG-PAC1 spheroids. Our analysis also indicates that YAP/TAZ target genes are upregulated together with myoepithelial markers. Importantly, siRNA targeting of TAZ expression in mSG-PAC1 spheroids diminished the expression of myoepithelial markers. Our results in this in vitro cell model implicate TAZ signaling in myoepithelial differentiation.
The integrin-mediated interaction of cells with components of the extracellular matrix (ECM) regulates many cellular processes including cell division. Cytokinesis is the last step of cell division and is critical for normal development and tissue homeostasis as it ensures the proper segregation of genetic and cytoplasmic material between daughter cells. Cytokinesis failure leads to defects in development and tissue differentiation, as well as tumorigenesis. Abscission of intercellular bridge that connects presumptive daughter cells is the last step of cell division. The mitotic kinesin-like protein 1 (MKLP1) plays a central role in positioning the abscission machinery. Here, we show that α6 integrins promote successful cytokinesis in salivary gland epithelial cells by regulating the expression of MKLP1. RNAi-mediated depletion of α6 integrins inhibits cytokinesis and the expression of MKLP1 and p90 ribosomal-S6kinase 2 (RSK2). Depletion of RSK2 results in similar defects in cytokinesis and also inhibits the expression of MKLP1, suggesting that the expression of RSK2 is required downstream of integrins to promote MKLP1 expression and successful cytokinesis. RNAi-mediated depletion of RSK2 in embryonic salivary glands in organ culture also results in the inhibition of cytokinesis and MKLP1 expression, indicating the physiological significance of this pathway.
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