Using both epithelial cells and monocytic cells, we have identified both similar and markedly different responses to exogenous PPIA. Several of the key new findings include the following: i) The biological activity of PPIA was partially abrogated by a PPIA active site mutant (Figure
2), thereby both confirming specificity and suggesting that the PPIA active site is important for some of its many biological activities. However, the important and potentially novel finding here is that the PPIA active site mutant still retains a significant residual biological activity, despite the fact that this mutant is reported to be only 1 % as enzymatically active as the wild-type [
32]. This suggests that the PPIase activity of PPIA may not be important for all of its extracellular functions, which is analogous to recent findings that have suggested the PPIase activity is not required for all of its intracellular functions [
45,
46]. ii) Similarities were identified in response to PPIA, which included the production of IL-8 and p21 (Figure
1) as well as activation of NFκB (Figure
3). In fact, a novel finding here is that the PPIA-mediated response for several cytokines is completely reliant on the downstream activity of NFκB and consistent with this, all cell lines also showed translocation of NFκB from the cytoplasm to the nucleus upon PPIA stimulation (Figure
3). These similarities may suggest that extracellular PPIA contributes to the relatively high concentrations of key factors such as IL-8 that drive cancer progression [
27] and p21 levels that have also been found to correlate to the progression of pancreatic cancer [
24]. iii) Several differential responses were identified in response to PPIA. These cell-dependent responses were observed in regard to PPIA stimulation of ERK1/2 phosphorylation, indicating that the signaling pathways that lead to NFκB translation are cell-dependent. Another novel finding here is that there is a differential response observed for cell lines even derived from the same cancer. Specifically, the two pancreatic cell lines, PANC-1 and L3.6pL, exhibit a markedly different response to extracellular PPIA with regard to IL-5 and IL-6 (Figure
1C,D) as well as ERK1/2 phosphorylation (Figure
4). Differential responses to exogenous PPIA also hold true for the monocytic cell lines used here as well, MOLM13 and U937 cells. Specifically, despite the common myeloid lineage of both cell lines, extracellular PPIA only induced cell surface expression of BSG within MOLM13 cells, but not in U937 cells (Figure
5B,C). Nonetheless, our identification of PPIA-induced cellular BSG expression has important implications for cancer progression in regard to the relationships between PPIA, BSG, and MMPs. Namely, our data show that in some cell types, PPIA stimulates cellular BSG expression, which is in turn a known regulator of many MMPs that drive tumorigenesis [
47]. iv) Extracellular PPIA was not necessarily reliant on cellular BSG for all of its activities (Figure
5). For example, knockdown of the cellular BSG in monocytic cells resulted in abrogation of PPIA-mediated ERK1/2 phosphorylation. However, knockdown of cellular BSG did not result in a detectable change in PPIA-mediated stimulation of IL-8, suggesting that BSG is not the sole cellular target of extracellular PPIA and, therefore, opening avenues for the further identification of PPIA cellular receptors.
The identification of cellular BSG as a target for extracellular PPIA has greatly enhanced the field of PPIA-mediated signaling [
6], yet studies such as those conducted here reveal that PPIA signaling is far more complex than previously thought. Specifically, extracellular PPIA likely interacts with multiple cellular receptors. For example, our previous atomic resolution investigations with PPIA and BSG revealed that these two proteins bind with millimolar affinity and therefore may require other mediators to enhance their biological interactions [
17]. This is supported by our studies here that have shown recombinant PPIA is biologically active at much lower concentrations that are within the micromolar range, which is more reflective of those concentrations that have been detected in vivo [
10]. More importantly, the very fact that BSG is necessary for PPIA stimulation of ERK1/2 phosphorylation (in MOLM13 cells in Figure
5A), but dispensable for IL-8 stimulation (in U937 cells in Figure
5C) could imply that there is at least one more cellular receptor beyond BSG. This is further supported by the differential cellular response imparted by extracellular PPIA (Figure
1 and Figure
4), which may not be surprising in retrospect. For example, intracellular PPIA interacts with numerous targets that include intracellular regions of receptors such as prolactin [
48] along with multiple kinases [
16]. Thus, extracellular PPIA has likely also evolved to interact with multiple extracellular targets thereby regulating many cellular responses that are simply dependent on expressed receptors. In fact, extracellular PPIA has recently been shown to mediate polymerization of the extracellular protein hensin [
49], which in turn mediates assembly of the extracellular matrix. Previous studies using PANC-1 cells have shown that PPIA-induction of IL-17 is only partially reduced upon blocking cellular BSG, also suggesting that other cellular receptors are involved in IL-17 stimulation [
19]. All of these findings have critical implications to a field where BSG has been thought to be the only cellular receptor. Unfortunately the identification of PPIA targets has been met with limited success (both extracellular and intracellular), which is likely due to the transient nature of PPIA interactions that makes simple pull-down experiments difficult. Future studies aimed at identifying the other cellular receptors of PPIA will likely require recently developed approaches that are more directed at low affinity interactions such as crosslinking methods [
50,
51]. Thus, hensin and BSG already provide an initial short list of the multiple proteins that extracellular PPIA engages, yet several studies including ours here also suggest that glycosaminoglycans or other potential cellular protein receptors serve as other cellular targets. Our confirmation that PPIA engages one such glycosaminoglycan directly through its active site, i.e., heparin (Figure
6), supports several previous studies that have indicated that these glycans serve as the initial interactions of extracellular cyclophilins with the cell surface [
1,
43,
44,
52].