Historically, substantial evidence has accumulated to implicate ErbB1, the cardinal member of the ErbB family, in the control of epidermal growth and differentiation. However, much of the earlier data made use of EGF binding assays that would not detect ErbB1 specifically [48
]. The discovery of multiple additional members of the ErbB family [1,2
] raises the question of whether ErbB1 is the sole mediator of these processes in the skin. We have addressed this question via a combination of approaches, including knockout mice, immunoblotting, pharmacologic inhibitors, and immunolocalization experiments.
As shown in , both PD153035 and PD158780 markedly inhibited the HB-EGF mRNA response in organ cultures prepared from wild type mice. Both compounds inhibited the mouse skin response with the same potency they displayed in human skin organ culture (Ref. [17
] and data not shown). This suggests that ErbB signaling plays a similar role in the skin organ culture response in man and mouse. However, two strains of ErbB1 (-/-) animals demonstrated very similar induction of HB-EGF mRNA compared to their wild-type littermates () or to other wild-type animals (). This would not be expected if ErbB1 were the only relevant ErbB species involved in the skin organ culture response. These results cannot be explained by nonspecificity of the inhibitors, as we utilized PD153035 and PD158780 at 0.2 to 1 µ
M, concentrations well below their thresholds of activity against non-ErbB RTKs (≈50 µ
]. Alternatively, it could be argued that the exon 2 knockout construct might express the cytoplasmic domain of ErbB1 due to alternative splicing. Indeed, a minor ErbB1-immunoreactive band has been reported in keratinocytes from another ErbB1 exon 2 knockout strain [49
]. However, the ErbB1 exon 1 knockout we tested has been shown to be totally lacking in ErbB1 mRNA expression [25
], and the only ErbB1 mRNA expressed by the exon 2 knockout we tested contains multiple stop codons (Z.W., unpublished data).
It could also be argued that ErbB family members other than ErbB1 come into play only when ErbB1 is genetically ablated. However, our own earlier studies of human skin organ culture [17
] argue against this interpretation. In those studies, mAb 225 IgG was utilized as a reagent to block inducible HB-EGF expression in organ culture. 225 IgG is known to specifically inhibit ligand activation of ErbB1-RTK in A431 cells [50
]. Despite use of 225 IgG concentrations 16 times higher than required to completely inhibit ligand-induced ErbB tyrosine phosphorylation, and >50 times higher than that required to maximally inhibit EGF-inducible TGF-α
], 225 IgG inhibition was substantially less complete than that produced by nontoxic doses of the pan-ErbB inhibitor PD153035 ( of this report, and of [17
]). As discussed in more detail below, documents robust expression of ErbB1, ErbB2, and ErbB3 in human skin. Taken together, these results strongly suggest that multiple ErbB species participate in the human skin organ culture HB-EGF response, even when ErbB1 is present.
In order to identify sensitive and specific reagents for detection of each ErbB species, we screened a total of 15 commercially available antibodies. To validate the specificity and sensitivity of these antibodies, we tested them against a panel of commonly used tumor cell lines. The results provide a uniform comparison of ErbB expression in these lines for the first time. MDA-MB-468 cells expressed large amounts of ErbB1, moderate amounts of ErbB3, and a small amount of ErbB2, as previously reported [51,52
]. In our hands, MCF-7 cells expressed ErbB2 and ErbB3, but no ErbB1 and little or no ErbB4 (). It has long been established that ErbB2 and ErbB3 are expressed by MCF-7 cells [53
]. While one recent report found expression of ErbB4 in this cell type [54
], two other reports found little or no ErbB4 unless it was ectopically expressed [55,56
]. A431 cells expressed substantial amounts of ErbB1, ErbB2, and ErbB3, but very little ErbB4 (). These findings were confirmed by immunoprecipitation using antibodies directed against different epitopes of ErbB1, ErbB2, and ErbB3 (). This pattern is similar to previous reports [57,58
] and is of interest because earlier studies emphasized gene amplification and overexpression of ErbB1 in A431 cells [42
] without considering the other ErbB species. We found that MDA-MB-453 cells expressed large amounts of ErbB2, lesser but substantial amounts of ErbB3 and ErbB4, and small amounts of a 180- to 185-kDa band detected by anti-ErbB1. This band probably represents cross reactivity against ErbB2 because it co-migrated with ErbB2 and because we could not detect it by immunoprecipitation using a different anti-ErbB1 antibody (). This expression pattern is also in good agreement with previous reports [45,52
]. However, we found more ErbB4 in MDA-MB-453 cells than reported by others [52
]. Substantial amounts of all four ErbB mRNAs have been detected in MDA-MB-453 cells by RT-PCR [59
]. Taken together, these results validate the specificity of the antibodies used, and demonstrate that these frequently used tumor cell lines display characteristic patterns of ErbB protein expression. However, they also suggest that these lines probably undergo stochastic or adaptive changes in ErbB expression as they are maintained in culture.
Despite the fact that A431 cells, NHK, and MDA-MB-453 cells all expressed substantial amounts of ErbB2 and ErbB3, very different patterns of ligand-stimulated tyrosine phosphorylation were observed between ErbB1-expressing A431 cells and NHK on one hand, and ErbB1-nonexpressing MDA-MB-453 cells on the other (). Some of these differences are explicable in light of current theories of receptor activation [4
], known patterns of ligand-receptor binding and intrinsic RTK activity [1,2
], and the ErbB expression patterns shown in . MDA-MB-453 cells showed only a slight increase in tyrosine phosphorylation in response to EGF, as compared to a 20- to 100-fold induction in response to heregulin (). Similar observations have been reported recently [52
]. This response probably reflects the lack of ErbB1 in MDA-MB-453 cells, coupled with the fact that EGF does not bind efficiently to any of the other ErbB species [1,2
]. In contrast, A431 cells and NHK showed only a slight increase in protein tyrosine phosphorylation in response to heregulin, as compared to a 20- to 100-fold increase in response to EGF ().
This lack of heregulin responsiveness is only partially explained by recalling the very low affinity of heregulin for ErbB1 [60
] coupled with lack of ErbB4 (). Coexpression of ErbB2 and ErbB3 in cells that normally lack ErbB receptors leads to robust tyrosine phosphorylation of ErbB2 and ErbB3 [61,62
]. NHK expressed only low levels of ErbB3 ( and B
), meaning that these cells could only form limited amounts of ErbB2-ErbB3 heterodimers to serve as a functional heregulin receptor. This could explain why NHKs were unresponsive to heregulin. However, low ErbB3 levels cannot explain why heregulin-dependent tyrosine phosphorylation was not observed in A431 cells, as both of these receptors are well expressed in A431 (). This paradox can be explained by the observation that ErbB2 was confined to the cytoplasm of A431 cells (), suggesting that ErbB2 and ErbB3 are unable to form a functional heregulin receptor on the surface of A431 cells. Interestingly, a similar phenomenon was observed in intact skin. ErbB1 retains a predominantly peripheral plasma membrane and cortical submembranous location throughout the epidermis, forming a “chicken wire” pattern (). In contrast, ErbB2 assumes a diffuse punctate distribution throughout the cytoplasm in the basal and lower suprabasal keratinocytes that comprise the proliferative compartment of the epidermis, but transitions to a discrete “chicken wire” pattern in the highly differentiated keratinocytes of the upper epidermal layers ().
Taken together, the absence of ErbB4, the lack of cell surface ErbB2, and the inactive nature of the ErbB3 RTK lead us to conclude that addition of EGF to rapidly proliferating A431 cells or NHK must primarily stimulate ErbB1-RTK activity (although later activation of intracellular ErbB2 cannot be ruled out). These findings support the use of A431 cells stimulated briefly with EGF as a selective assay for ErbB1- RTK activity. Because we and others have found no ErbB1 in MDA-MB-453 cells, we can also justify the use of MDA-MB-453 cells stimulated briefly with heregulin as an assay for ErbB-RTKs other than ErbB1. Using these assays (), we found that the RTK inhibitor PD166547 was about 45 times more potent in EGF-stimulated A431 cells than it was in heregulin-stimulated MDA-MB-453 cells, confirming earlier reports of a 40-fold ErbB1 selectivity for this compound [43
]. In contrast, PD153035 and PD158780 were of very similar potency in the two assays and therefore represented “pan-ErbB” inhibitors. By comparing the potency of the three inhibitors in EGF-stimulated A431 cells, we could obtain an estimate of their intrinsic potencies. As shown in , PD166547 was at least four times less potent than either PD153035 or PD158780. PD166547 was also five to seven times less potent as a growth inhibitor for NHK, compared to the two pan-ErbB inhibitors (). These differences in growth-inhibitory potency approximately match the differences in intrinsic potency, despite the fact that PD166547 is 40 to 45 times more potent against ErbB1-RTK than against the other ErbB species. Taken together, these results strongly suggest that ErbB1-RTK plays a predominant role in the regulation of NHK growth in culture.
NHK growth inhibition required 50 to 100 times more RTK inhibitor than was required to inhibit ligand-stimulated ErbB tyrosine phosphorylation (). Similar observations have been made previously [44,45
]. Given the presence of the non-EGF mitogens insulin and bovine pituitary extract in the keratinocyte growth medium, this difference is most likely due to the contributions of non-ErbB signaling pathways to the process of keratinocyte growth. Indeed, we have observed that ErbB-RTKIs are at least an order of magnitude more potent as growth inhibitors when non-EGF mitogens are omitted from the growth medium [70
]. This phenomenon probably also explains the lower potencies of these compounds in the skin organ culture system (), in which a variety of signaling pathways in addition to ErbB are likely to be active.
These results are consistent with our previous work demonstrating that the mAb 225 IgG, which specifically blocks the ligand-inducible activation of ErbB1-RTK [50
], is a potent inhibitor of NHK growth [47,63
]. NHKs express high levels of the ErbB1-selective, EGF-like ligands HB-EGF, AR, and TGF-µ
in an autocrine fashion [46,47,64
]. In contrast, heregulin expression appears to be limited relative to TGF-µ
, AR, and HB-EGF in cultured keratinocytes [65
], and heregulins are not potent mitogens for keratinocytes relative to EGF [30,66
]. Moreover, heregulin produced only a slight increase in tyrosine phosphorylation in NHK () due to low expression of ErbB3 () and/or to intracellular compartmentalization of ErbB2 (). Taken together, these findings suggest that ligand-dependent ErbB-mediated keratinocyte proliferative responses pass primarily through ErbB1, whereas ligands of the heregulin family interacting with ErbB2 on the cell surface of upper-layer keratinocytes may play an important role in terminal differentiation. Recent studies (Predd H, Underwood R, Peterson T, Cook P, and Piepkorn M. Society for Investigative Dermatology 2001 Annual Meeting, Abstract 104) demonstrate that differentiating keratinocytes in culture and in intact human skin express heregulin in the same distribution we have observed for ErbB2. As ErbB1 expression is maintained throughout the epidermis (), ErbB1-selective ligands may also interact with ErbB2 in differentiated keratinocytes. Indeed, AR, which binds exclusively to ErbB1 [1,2
], has been implicated in the control of what is arguably the most highly differentiated response of keratinocytes: maintenance of the stratum corneum permeability barrier [67
This conclusion returns us to the original observations that initiated this work: mouse skin organ cultures appear to be highly dependent on ErbB signaling (), yet independent of ErbB1 (). Early epidermal wound healing responses are dominated by specialized and accelerated differentiation responses, which proceed for 1 to 3 days prior to the onset of significant epidermal proliferation in vivo
] and in organ culture [17
]. The primary function of these early responses, during which keratinocytes flatten and rapidly migrate to cover the wound, is to restore epidermal barrier function as rapidly as possible. Based on our results, we would speculate that signaling through ErbB family members other than ErbB1 in differentiated keratinocytes of the upper epidermal layers may be critical for these early wound healing responses, including induction of HB-EGF.
Finally, our data suggest that intracellular sequestration of ErbB2 may be an important mechanism by which malignant A431 cells limit their responsiveness to heregulin. If the interactions of ligands such as AR and heregulin with cell surface ErbB2 lead to growth arrest and terminal differentiation, as has been suggested for skin [67
] and mammary epithelial cells [69
], then the ability of A431 cells to limit cell surface expression of ErbB2 may be an important mechanism by which A431 and other skin carcinomas evade terminal differentiation signals in vivo
. The intermingling of differentiation and proliferation has long been a puzzling feature of squamous cell carcinomas. Unraveling the mechanisms by which A431 cells limit cell surface expression of ErbB2 may provide new avenues for differentiation therapy of epithelial cancers, which in aggregate account for over 90% of all human malignancies.