In the current report, we provide evidence that TGFα triggers HKCs to secrete hsp90α and that extracellularly located hsp90α in turn acts as a promotility factor for all major human skin cell types. Among the major cell types in skin, only HKCs secrete hsp90α. Mechanistically, TGFα triggers rapid membrane translocation of hsp90α and its secretion to extracellular environment via the exosome pathway. Among the four domains of hsp90α, only the middle domain (plus the charged sequence) is able to fully duplicate the promotility effect of the full-length hsp90α, independently of its ATP-binding and ATPase functions. Furthermore, extracellular hsp90α promotes migration of both epidermal and dermal cells though the cell surface receptor LRP-1/CD91. This report presents the physiologically significant finding that the extracellular hsp90α-induced skin cell migration could no longer be inhibited by TGFβ, which is abundantly present in skin wounds. TGFβ potently inhibits canonical growth factor-stimulated migration of DFs and HDMECs; therefore, our finding provides for the first time an explanation of how the dermal cells move into the wound bed in the presence of TGFβ. A schematic representation of these findings is shown in Fig. . In support of this model in vivo, we recently reported that topical application of purified hsp90α enhanced skin wound healing in nude mice (24
). Taking all of these results together, we propose that following wounding, increased TGFα from the serum stimulates HKCs to secrete hsp90α. After reaching its threshold, extracellular hsp90α not only promotes HKC migration for reepithelialization but also recruits dermal cells to migrate into the wound for new connective tissue formation, new blood vessel formation, and wound remodeling. Moreover, hsp90α's TGFβ-insensitive signaling has a clear advantage over that of the canonical growth factors in promotion of cell migration under the conditions of the TGFβ-rich microenvironment of skin wounds. Considering the fact that TGFβ potently inhibits dermal cell migration, such as that of DFs and HDMECs (3
), extracellular hsp90α may represent a bona fide physiological driving force for dermal cell migration into the wound bed to participate in wound healing.
FIG. 11. A schematic summary: the TGFα → hsp90α secretion → skin cell migration → wound healing model. Following skin injury, paracrine- or autocrine-released TGFα stimulates membrane translocation and secretion (more ...)
Historically, the hsp90 family chaperons regulate folding, transport, maturation, and degradation of a diverse, but selective, set of client proteins, in particular, signaling molecules (33
). Recently, extracellularly located hsp90 proteins have also been reported. Liao and colleagues showed that oxidative stress causes sustained release of hsp90α, which in turn stimulates activation of ERK1/2
in rat vascular smooth muscle cells (26
). Jay and his colleagues identified hsp90α, but not hsp90β, in the conditioned media of tumor cells (11
). Moreover, two groups showed that heat stress causes increased secretion of hsp90 and hsp70 (8
). Yu and colleagues showed that γ radiation stimulates secretion of hsp90β into conditioned medium in a p53-dependent manner (51
). As previously mentioned, these proteins have to undergo the exosome-mediated exocytosis that results in export of cellular proteins that lack a signal sequence. In contrast, these proteins cannot be secreted via the conventional endoplasmic reticulum/Golgi transport pathway. Exosomes, also called “intraluminal vesicles,” are contained within multivesicular bodies (MVBs), whose known function is to act as an intermediate in the degradation of proteins internalized from the cell surface or sorted from the trans-Golgi organelle (10
). However, MVBs and their exosomes can also fuse with the plasma membrane to release cargo proteins into the extracellular space (14
). In fact, all of the proteins that have been identified in exosomes are located in the cell cytosol or endosomal compartments and never in the endoplasmic reticulum, Golgi apparatus, mitochondria, or nucleus (44
). Hsp90α has been identified in exosomes (8
). In this paper, our data link EGFR activation to the exosome pathway, leading to membrane translocation and secretion of hsp90α. How EGFR communicates with the MVB protein-trafficking pathway remains an unanswered question.
How does extracellular hsp90α promote cell migration? Jay and his colleagues showed that extracellular hsp90α, but not hsp90β, interacts with and acts as an activator of MMP2 and that functional inhibition of extracellular hsp90α inhibited tumor invasion (11
). However, unlike the results seen with hsp90α, which is able to duplicate the full promotility effect of TGFα on HKCs, there is no evidence that the addition of any recombinant MMP alone can promote cell motility. In addition, the presence of MMP inhibitors, such as GM6001 or MMP Inhibitor III, had little inhibitory effect on hsp90α-stimulated HKC migration (unpublished results). Instead, we provided evidence that CD91 is the cell surface receptor that mediates hsp90α signaling to promote cell migration. CD91 is found in monocytes, hepatocytes, fibroblasts, and keratinocytes (19
). We showed that the three major skin cell types, HKCs, DFs, and HDMECs, were all CD91 positive and that all responded to the promotility effect of hsp90α. Basu and colleagues have shown that released hsp90α binds to CD91, triggering macrophages to secrete cytokines and causing dendritic cells to express antigen-presenting and costimulatory molecules (4
). In addition to hsp90α, other extracellular heat shock proteins, including gp96, hsp60, hsp70, and calreticulin, bind CD91 (5
). However, while CD91 is a common receptor for heat shock proteins, not every CD91-binding heat shock protein was able to stimulate cell migration. In our hands, for instance, recombinant calreticulin showed no detectable promotility effect on HKCs, which contrasts with the results seen with hsp90α or TGFα (unpublished data). Therefore, the outcomes of CD91 activation may depend upon binding to its 515-kDa extracellular domain by specific heat shock proteins, such as hsp70 for antigen presentation (6
) and hsp90α for cell migration. Interestingly, RNAi down-regulation of CD91 did not block TGFα-stimulated HKC migration in vitro (C.-F. Cheng and W. Li, unpublished data), again supporting our hypothesis that HKC-secreted hsp90α is physiologically important for recruiting dermal cells into wounds.
Hsp90α is composed of an N′-terminal domain, a charged sequence (linker), a middle domain, and a C′-terminal domain. Monoclonal antibody screening analyses suggest that the middle and C′-terminal domains are exposed at the surface of hsp90α protein (34
). Analysis of the crystal structures of yeast and Escherichia coli
hsp90 revealed that the middle domain forms a highly conserved surface loop, suggesting a common role as a potential client protein binding site (2
). Our study showed that the combination of the middle domain plus the changed sequence strongly binds CD91 and exerts the same full-promotility effect as full-length hsp90α. Confirmation of these findings in in vivo wound healing models could further reveal the therapeutic potential of hsp90α.
Finally, the observation that TGFα selectively stimulates secretion of hsp90α in HKCs, but not in DFs, was unexpected, especially since TGFα can promote DF migration as well (23
). This finding suggests a previously unrecognized signaling mechanism: a single growth factor can use different mechanisms in two geographically close skin cell types to induce the same cellular response, i.e., motility. While the physiological implication of such TGFα signaling specificity remains to be studied, it is conceivable that a possible “linker” for communication between TGFα-bound EGFR and hsp90α-containing exosomes is selectively present in HKCs but absent in the dermal cell types. The nature of such a linker could be one of several possibilities by which intracellular signaling molecules interpret upstream signals. First, the signaling molecules can be differentially organized by scaffolding proteins, resulting in a variety of combinations. Second, distinct locations of the same signaling molecules can dictate specific biological responses. Third, the strength and duration of a given signal/pathway can affect the outcomes of the responses. The choice of these mechanisms and their responses may differ from cell type to cell type (37
). The bottom line is that differences in the cellular context of HKCs and DFs are responsible for determining the signaling specificity of TGFα.