Figure 6

Muc2, but not Muc5ac, secretion increases with pro-inflammatory stimulation in GPTE cells.a) Total protein concentration of pooled samples of mucin collected from the apical surface or as intracellular lysates of non-stimulated GPTE cell cultures was determined using the Bradford assay. Equal amounts of protein were used to coat ELISA wells (76 ng/well), and then the presence of Muc2 or Muc5ac was examined. Results indicate that, in unstimulated GPTE cells, there is a higher ratio of intracellular to secreted Muc2 when compared to the ratio of intracellular to secreted Muc5ac. Samples from six cultures were pooled. Error bars represent assay variance of triplicate measurements. b) GPTE cells were exposed to air for 7 days during which time goblet cell differentiation occurred and mucus secretion began. Apical surfaces were washed, media changed, and then collected for secreted mucus baseline after 12 hrs incubation at 37°C, 5% CO₂. Media was then changed and cultures were rested for 8 hrs and exposed to cytomix for 4 additional hrs. Relative amounts of Muc2 or Muc5ac in experimental and baseline collections were determined by ELISA. Experimental absorbance values were then corrected with baseline values, allowing each well to serve as its own control. These normalized values are expressed as a percentage of CONSTITUTIVE secretion observed during the 12-hr rest/exposure period, with the amount of Muc2 or Muc5ac secreted in cultures exposed only to media arbitrarily set at 100% of constitutive secretion. Results indicate Muc2 secretion is augmented by cytomix exposure, while Muc5ac secretion is equivalent in cytomix-treated and media-exposed (control) cultures. n = 5 in each group. ** = Significant change from constitutive mucin secretion by Student's t-test (p < 0.05). c) GPTE cells were exposed to cytomix or media (as a control) for 4 hrs. Then cells were lysed and assayed for intracellular mucin. The amount of Muc2 or Muc5ac in intracellular lysates from cells exposed only to media was arbitrarily set at 100%. In intracellular lysates from cells exposed to cytomix, both Muc2 and Muc5ac production was increased 50% over CONSTITUTIVE mucin levels. Subsequent time points (8 or 12 hrs exposure) showed a return to constitutive intracellular levels (data not shown). n = 6 in each group. * = Significant change from constitutive mucin production by Student's t-test (p < 0.01). d) GPTE cultures were exposed to 4, 8, and 12 hrs of cytomix. Total RNA was collected and Muc2 and Muc5ac transcript levels were determined using real-time RT-PCR. At 4 hrs, Muc2 mRNA levels were significantly increased when compared to constitutive expression. A trend toward an increase in both Muc2 and Muc5ac mRNA levels following cytomix exposure when compared to constitutive expression was observed at later time points. n = 4–6 in each group. * = Significant change from constitutive mucin production by Student's t-test (p < 0.05). All data points are presented as mean ± SEM.