In this study we provide evidence for a synergistic upregulation of SP-B mRNA in type II pneumocytes induced by a combined treatment with glucocorticoids and caffeine. We show that this increase of SP-B mRNA is mediated by an intracellular enrichment of cAMP and is associated with an upregulation of the SP-B transcription cofactor ErbB4 as well as the GR. This synergistic activity mainly affects SP-B mRNA levels and increases mature SP-B, but has, at least in H441 cells, no further impact on the caffeine-mediated increase of total SP-B translation.
An induction of SP-B mRNA by dexamethasone and other glucocorticoids has already been described 
and could be confirmed in this study. The mechanisms leading to an increase of SP mRNA transcription by glucocorticoids have been already widely discussed and several transcription factors as well as the GR have been implicated in this upregulation 
. It has also been shown that SP-B mRNA accumulation in H441 cells and explants is a complex interplay between SP-B gene transcription and modification of SP-B mRNA stability and that posttranscriptional factors also influence SP-B expression 
Compared to glucocorticoids, we found a slight, dose dependent induction of SP-B mRNA in H441 cells by caffeine alone, which has not been described so far. In combination with dexamethasone and cAMP, isobutylmethylxanthine has been widely used to preserve the phenotypic characteristics of type II epithelial cells in cell culture 
, although an exclusive impact of methylxanthines on the expression of SPs is not well described yet. Considering the ability of caffeine, like many other methylxanthines, to unspecifically inhibit the activity of PDEs, it seems plausible that the observed induction of SP-B mRNA expression by caffeine is mediated by an intracellular enrichment of cAMP, since this effect could be only observed, if high concentrations of caffeine were used. In fact, the observed 10-fold higher level of SP-B mRNA in H441 cells achieved after the exposure to cAMP alone is in consistence with this assumption and with the so far reported role of cAMP as inducer of SP-B mRNA expression 
and enhancer of GR DNA-binding via protein kinase A 
To address, whether the combined treatment of caffeine and glucocorticoids has any further effect on transcription of SP-B mRNA rather than simple additive or reductive effects, we investigated this combined treatment by measuring SP-B mRNA levels. We found, that all metabolic active glucocorticoids induce a strong synergistic upregulation of SP-B mRNA if combined with caffeine. Combinations of dexamethasone, cAMP and isobutylmethylxanthine, a competitive non-selective PDE inhibitor 
, have been described to facilitate differentiation of type II cells from precursor lung epithelial cells and to synergistically induce the transcription of a various set of genes, including SP-A, SP-B and SP-C 
. By combination of various glucocorticoids with caffeine rather than with cAMP, we could show a comparable synergistic induction of SP-B mRNA as previously observed in cultured human fetal lung epithelial cells using dexamethasone, cAMP and isobutylmethylxanthine 
. This may indicate a common upregulatory mechanism of caffeine, cAMP and PDE inhibition. By substituting caffeine by cAMP in combination with dexamethasone and without any additional PDE inhibitors we could mimic the previously reported synergistic mRNA induction 
to the same extend as in combination with caffeine, indicating an essential mediating role of cAMP in this process.
We found that caffeine contributes to the synergism in SP-B mRNA induction only at high, non-physiological doses. Supposing the exclusive role of caffeine as unspecific PDE inhibitor in the context of the above described synergism, a comparable inhibition of PDEs using physiological doses of inhibitors could avoid the usage of high doses of caffeine, thereby eliminating undesirable side effects but maintaining the synergistic mRNA induction, which then could be of benefit in surfactant homeostasis. For this purpose we used the PDE inhibitor rolipram, which is a first generation PDE4 inhibitor characterized by anti-inflammatory and anti-immunomodulatory effects 
. In fact, a dose dependent, significant increase in SP-B mRNA induction was observed when caffeine was substituted by rolipram. The use of selective PDE inhibitors of the second generation such as the PDE4 inhibitors cilomilast and roflumilast, exhibiting longer half-lives and fewer side effects, may further increase intracellular cAMP levels and thus also potentially therapeutic SP-B levels 
. The therapeutic potential of PDE4 inhibitors was further demonstrated by their administration to different animal models with lung diseases, in whom they have been shown to improve lung alveolarisation 
and to attenuate persistent lung injury 
To address, which mechanisms are directly responsible for the observed SP-B mRNA upregulation induced by caffeine, we first measured the mRNA levels of TTF-1, the most important transcription factor involved in SP mRNA transcription 
. However, the reported induction of TTF-1 mRNA expression by dexamethasone and cAMP 
could be confirmed by qPCR only in A549, but not H441 cells after the treatment with caffeine instead of cAMP. A possible explanation for this disagreement could be the accessibility to different regulatory mechanisms leading to SP gene transcription in fetal or different lung epithelial cell subtypes versus H441 cells, which are derived from adult papillary adenocarcinoma cells, since it is known that other factors play a central role in cell specific activation of SP genes 
. The hypothesis that glucocorticoids restore TTF-1 and SP-B levels by increasing the TTF-1 autoregulatory mechanism only in hypoplastic fetal lungs supports this possibility 
. Since it is known that cAMP is able to mediate conformational changes in protein kinase A, which lead to the phosphorylation of TTF-1 
, an involvement of TTF-1 in the observed SP-B mRNA induction in H441 cells could also be explained by its phosphorylation rather than by its upregulation.
One other recently identified transcriptional cofactor of the SP-B gene is ErbB4 
. It has been already associated with the induction of fetal surfactant phospholipid synthesis 
and seems to have a crucial regulatory role in fetal lung development 
. We found that caffeine, but not dexamethasone, is able to induce an increase in ErbB4 mRNA in H441 cells and in A549 cells. Therefore, ErbB4 may contribute to the observed caffeine-induced expression of SP-B mRNA and its synergistic induction in combination with dexamethasone. The examination of other SP-B transcription factor mRNAs like Jun B, c-Jun, SP1, SP3, and HNF-3α indicated no involvement in the described synergism.
To directly examine the influence of SP-B transcription factors, we performed SP-B promoter analyses. By this, an approximately 900 bp promoter-region of human SP-B investigated in luciferase assays was not reflecting the same synergistic effects for the reporter gene. These results are indicating a more complex regulation of SP-B mRNA transcription initiation, which may include other factors like cis- or trans-activating elements in combination. Additionally, an increase of SP-B mRNA stability is neither be detectable by the investigation of the SP-B promoter-region. For glucocorticoids this has been previously shown using also H441 cells, where it was postulated that the influence of glucocorticoids on SP-B mRNA expression is more likely due to changes in mRNA stability rather than enhanced transcription 
Besides influencing SP-B transcription factors, a modification of the glucocorticoid signal transduction pathway by caffeine could be an explanation for the observed synergistic induction of SP-B mRNA expression by caffeine and glucocorticoids, since it is known that the action of glucocorticoids is mediated by the GR 
. We found a slight upregulation of GR mRNA after induction with caffeine, which was not modified by the addition of dexamethasone. Some authors already reported an upregulation of both GR mRNA and protein levels by glucocorticoids in various cell lines of lymphoid leukemias 
and a caffeine-mediated enhancement of GR activity in human osteoblastic cells 
. Flow cytometric measurement of GR protein levels revealed a downregulation of the GR by dexamethasone, which could be partly restored by the addition of caffeine. This would indicate that caffeine influences the ability of glucocorticoids to increase SP-B mRNA levels by modifying GR expression and therefore possibly reversing the same inhibitory effects of glucocorticoids on SP-B mRNA transcription as shown for SP-A mRNA 
. Therefore, reversing this inhibitory effect on transcription, the enhanced mRNA stability mediated by glucocorticoids 
could lead to enormous new amounts of SP-B mRNA, which explains the synergistic induction reported here.
The enhancement of SP-B mRNA by glucocorticoids alone and the synergistic induction by dexamethasone in combination with caffeine was completely abolished by the addition of the GR-inhibitor RU486, which indicates a major involvement of the GR signaling pathway.
When investigating the induction of SP-B by caffeine and glucocorticoids at the protein level, different results were obtained, depending on whether mature or total SP-B (including mature and un- or partially processed forms of SP-B) was investigated. If the mature form of SP-B was analysed by immunoblotting, we found these fully processed form induced by dexamethasone and also significantly to a larger extend by the combination of dexamethasone and caffeine, while no mature SP-B could be detected in untreated cells and cells treated with caffeine alone. This is also in accordance with the observation of previous studies, which claim that SP-B is not fully processed in untreated H441 cells 
. It also further indicates that parts of the cellular machinery responsible for the final processing of pro-SP-B are inducible by glucocorticoids and, to a higher extent, by their combination with caffeine in H441 cells. When the content of total SP-B was investigated by immunofluorescence and flow cytometry, only a slight but marked increase was obtained by the induction with caffeine. The pro-forms of SP-B are abundantly expressed in H441 cells and may therefore represent static forms which may be affected differently or not affected at all by glucocorticoids in combination with caffeine. Changes of mature SP-B were masked by the detection of the pro-SP-B forms in immunofluorescence stainings. We therefore speculate that caffeine-induced inhibition of PDE and the subsequent increase of cellular cAMP levels are only influencing SP-B mRNA transcription and processing of pro-SP-B forms, but have no impact on caffeine-mediated increase of SP-B translation.
In conclusion, we described a synergistic upregulation of SP-B mRNA in H441 and A549 cells induced by glucocorticoids in combination with caffeine, which was associated with increased levels of the GR and of the SP-B transcription cofactor ErbB4. Our results indicate that basic SP-B mRNA transcription initiated by glucocorticoids can be potentiated via an increase of cellular cAMP, achieved by a caffeine-mediated inhibition of PDEs. These findings may contribute to our understanding of how the expression of SP genes is regulated. Therefore, the administration of caffeine in combination with glucocorticoids may be of benefit in surfactant homeostasis during the treatment of preterm infants.