Msx2 is an important transcription factor that regulates the development of various organs. In humans, Msx2 gene localizes to chromosome 5 and a mutation in the homeodomain causes craniosynostosis, the premature fusion of calvarial sutures and a common developmental anomaly that causes abnormal skull shape 27
. In developing human fetal skin, Msx2 expression patterns were detected throughout the epidermis and dermis, with the greatest level of expression in the suprabasal epithelial layer. In the adult human skin, Msx2 was confined to the basal and suprabasal layers of the epidermis, with the strongest expression in the hair follicles 9
and was required, with Foxn1, to maintain Notch1 expression in the hair follicle matrix 25, 28
. In this study, we found that Msx2 is transiently up-regulated during healing of the excisional wounds of normal mouse skin and its expression coincides with the migratory pattern of epithelial cells. In addition, Msx2 null mice demonstrate faster re-epithelialization and wound closure in comparison to their WT littermate controls. When wound healing is compared at the cellular level, differences in keratinocyte differentiation is noted. At the microscopic level, collagen, SMA, and tenacin C are elevated in granulation tissue of Msx2 null mice compared to that of same stage WT controls. In vitro assays reveal a faster keratinocyte migration by Msx2 null keratinocytes and an increased ability in collagen matrix contraction by Msx2 null fibroblasts. Therefore, the results suggest that Msx2 regulate cellular competence of keratinocytes and fibroblasts in skin injury repair.
Injury repair is one of the most complex biological processes. After an injury, multiple biological pathways immediately become activated and are synchronized to respond. In the postnatal stage, the process commonly leads to a non-functioning mass of fibrotic tissue known as a scar. By contrast, early in gestation, injured fetal tissues can heal in a regenerative fashion without scar formation 1
. These differences in response to the same stimuli may be explained by the developmentally regulated general make up of the participating soluble and cellular and extracellular matrix components 15
. More importantly, however, is the intrinsic difference in the composition of sub-cellular ingredients that govern cellular response or competence. The competence is molecularly based on the makeup of different cell membrane receptors, intracellular signaling molecules, transcription factors and chromatin modifications. The homeobox transcription factors such as Msx1 and Msx2 are candidates of such molecules in which they differentially expressed by cells of different developmental stages and in a variety of locations involved typically in epithelial-mesenchymal interactions during organ morphogenesis 27, 29, 30
. Furthermore, Msx2 is expressed in developing epidermis and hair follicles in mouse fetus while limited to the hair follicle in adult skin 31
. As shown in this study, Msx2 is transiently induced in the epidermal cells upon wounding, beginning at PWD 1, reaching higher level by PWD 3-5, dissipating by PWD 7, and is completely absent from the epidermis by PWD 14 (). Interestingly, the expression seems to start from the infundibulum region of the hair follicles surrounding the wound, and appears to migrate towards the wound as shown by the image of a centripetally arranged blue tracks. This is reminiscent of similar tracks observed in the wounding of Tg(Krt15-cre/PGR)22Cot;R26R 32, 33
. It is also consistent with the thought that some cells derived from infundibulum regions of hair follicles emigrate to the epidermis 34, 35
. While the results of these previous investigations suggest that cells from hair follicles migrate out to aid in the process of re-epitheliazation during skin wound repair, Ito et al. 32, 33
observed that the bulge derived cells are there only for a few weeks, and eventually disappear from the epidermis. On the other hand, Morgan et al.34, 35
, using ShhGFPcre;R26R
mice, showed that some cells in the upper follicles indeed stay in the epidermis. Collectively, these studies point out to the possible multiple origins of cells in the re-epitheliazation process, i.e., from the proliferation of the transiently amplifying cells in the basal layer, activated stem cell in the basal layer, de-differentiation of suprabasal keratinocytes, both follicle bulge and non-bulge cells, or the combination of some of these possibilities 36
. Based on the pattern of Msx2 expression in our study, we speculate that the Msx2 positive epidermal cells from the infundibulum of hair follicles are activated, migrate to the wound, and participate in re-epithelialization ().
A balanced homeostasis during the healing process is required among proliferation, differentiation, apoptosis and migration to achieve the formation of a new epithelium that covers the wound. Since the re-conditioning of cell behavior from physiological homeostasis to active re-epithelialization requires epigenetic changes that involves homeobox genes, the accelerated closure of excision, full thickness wound in the dorsal skin of Msx2 null mice () may be contributed by an increase in keratinocyte migration and differentiation (). Our in vitro migration assay supports the altered/increased migratory phenotype of Msx2 null keratinocytes (). Consistently, past findings from our laboratory have shown that an overexpression of Msx2 seems to have an opposing effect on keratinocytes differentiation with increasing cell proliferation in the suprabasal keratinocytes 8, 31, 37
. Interestingly, mice overexpressing Msx2 also exhibit a thicker epidermis accompanied with more hair follicles and hypercellularity, while a lack of Msx2 led to a thinner dermis and ‘cyclic alopecia’, or an asynchronous cycling of hair follicles, and defective hair shaft differentiation 25, 31
. Therefore, in the current study we conclude that, when faced with the same wound stimuli, keratinocytes without Msx2 may undergo accelerated differentiation; while keratinocytes with Msx2 stay longer in the morphogenetically malleable progenitor stage and allow time for regeneration.
Also in the current study, unlike the epidermal cells, Msx2 is not induced in fibroblasts during skin wound healing (). Nonetheless, for the Msx2 null mice we observe an increase of SMA in the wound fibroblasts and tenacin C in the extra-cellular matrix of granulation tissue (). It is likely that the effect may be mediated by some factors (co-modulators or targets of Msx2) secreted by the epidermis, highlighting the role of Msx genes in epithelial-mesenchymal interactions 38
. Therefore, the absence of Msx2 may alter the microenvironment around the wound and cause a change in the overall pattern of wound healing. As more functional studies are carried out with different homeobox genes, we should gain more insight on the molecular basis of cell competence in response to injuries. For instance, some of the more recent studies have shown that Hoxd3 enhances cell motility, increase the expression of integrin α5β1 and αvβ3 but decrease that of E-cadherin in normal mouse wounds 39
; while under diabetic situation, Hoxd3 can accelerate wound healing by inducing collagen synthesis 40
. Hoxb4, on the other hand, increases cell proliferation and reduces the expression of α2 integrin 41
. Mice deficient of Prx2
, a homeobox gene preferentially expressed in mesenchyme, exhibit altered healing of fetal wounds but not post-natal skin wounds 9, 42
. Pou2f3 inhibits keratinocytes differentiation induced by wounding 43
. Since wound repair recapitulates embryonic morphogenesis, Hox
genes are clearly a significant player in controlling cellular competency in post-natal tissue injury repair. Certainly more in depth and cohesive investigations are required to gain insights in the difference among healing of individuals of various age group and disease conditions.
In summary, consistent with the notion suggested in earlier correlated studies 9
, this study presents an example in which the presence or absence of a homeobox gene can affect the course of wound healing by altering the timing of proliferation and differentiation. The study also pointed out that Msx2 expression may be important for the homeostasis of proliferation and maturation process of basal keratinocytes in the inter-follicular epidermis and subsequently mesenchymal activities in the healing process. The understanding of these intrinsic cellular differences is fundamental to the eventual development of novel therapeutics for wound healing by modulating molecules constituting cellular competence.