Heidi Koegel's research while affiliated with Eawag: Das Wasserforschungs-Institut des ETH-Bereichs and other places

Wound healing in hpm mice. (A) Morphometrical analysis of wound healing parameters of 5 day wounds. Slightly reduced wound closure in hpm mice (control: n = 19, N = 7; hpm: n = 21, N = 7). The area of the hyperproliferative epithelium (HE) was similar in 5 day wounds of control and hpm mice (control: n = 14, N = 6; hpm: n = 11, N = 6). (B) Morphometrical analysis of wound healing parameters 13 days after wounding. The wound width was significantly increased (p = 0.0005; control: n = 16, N = 5; hpm: n = 16, N = 5) and the epidermis still hyperthickened in 13 day wounds of hpm mice (p = 0.0013; control: n = 19, N = 7; hpm: n = 21, N = 7). (C) 13 day wounds were examined for lacZ expression. Black arrowheads indicate the edges of the wound, white arrowheads mark an area of lacZ negative keratinocytes in the middle of the wound epithelium (control: N = 3; hpm: N = 4). Boxed area is shown at higher magnification. n, number of measurements; N, number of mice. (6.50 MB TIF)

Targeting strategy for the hpm allele. (A) Partial map of the β1A wild-type (wt), the hpm knock-in allele before (hpmKIneo-tk+) and after Cre-mediated deletion of the floxed neo-tk cassette (hpmKIlox). External probes used for Southern blotting after BamHI digest and genotyping PCR primers are indicated. Cyto-cDNA, a wt 141 bp cDNA fragment coding for the complete cytoplasmic domain of the β1A integrin gene including the endogenous stop codon; E15cyto, endogenous sequence of exon 15 encoding for the membrane proximal part of the β1 integrin cytoplasmic tail; filled boxes, exons; D, exon D; neo, neomycin resistance gene; tk, thymidine kinase gene; triangles, loxP sites; pA, polyadenylation signal. (B) Left panel: Southern blot analysis with the 5′external probe demonstrating recombination of the targeting construct (hpmneo-tk) into the wt β1 integrin locus giving rise to a 22.9 kb wt band and a 7 kb recombinant band. Right panel: Southern blot analysis with the 3′external probe demonstrating the deletion of the neo-tk cassette from the knockin hpmKIneo-tk+ allele (hpmKIlox) with a 22.9 kb wt band and 16 kb hpmKIlox band. (C) Representative PCR on genomic DNA from tail snips of offspring from hpmKIlox/+ intercrosses using a forward primer (primer S) binding to the transmembrane coding sequence of the β1 integrin gene, within exon 15 and a reverse primer (primer AS) binding to an intronic sequence downstream of exon 15. The 423 and 191 bp DNA bands correspond to the hpmKIlox and the wt β1 integrin allele, respectively. (0.95 MB TIF)

Integrin profile of hpm keratinocytes. Cell surface expression of integrins in freshly isolated keratinocytes from control and hpm mice at indicated ages were assessed by flow cytometry. Keratinocytes were stained with antibodies against α2, β4 and α6 integrins. Expression of integrin subunits was normalized to the expression level of age-matched controls. Cell surface expression of α2 integrin is significantly reduced in 3-week-old hpm mice and increases to control levels in 20-month-old hpm mice. Expression of α6 and β4 integrin in young and old hpm mutant mice is increased compared to controls (middle and lower panel, respectively). Mean fluorescence intensities were corrected for background fluorescence. Error bars indicate s.d. At least 2 control and 3 hpm mutant mice per developmental stage were analysed. (0.90 MB TIF)

The transcription factor serum response factor (SRF) plays a crucial role in the development of several organs. However, its role in the skin has not been explored. Here, we show that keratinocytes in normal human and mouse skin expressed high levels of SRF but that SRF expression was strongly downregulated in the hyperproliferative epidermis of wounded and psoriatic skin. Keratinocyte-specific deletion within the mouse SRF locus during embryonic development caused edema and skin blistering, and all animals died in utero. Postnatal loss of mouse SRF in keratinocytes resulted in the development of psoriasis-like skin lesions. These lesions were characterized by inflammation, hyperproliferation, and abnormal differentiation of keratinocytes as well as by disruption of the actin cytoskeleton. Ultrastructural analysis revealed markedly reduced cell-cell and cell-matrix contacts and loss of cell compaction in all epidermal layers. siRNA-mediated knockdown of SRF in primary human keratinocytes revealed that the cytoskeletal abnormalities and adhesion defects were a direct consequence of the loss of SRF. In contrast, the hyperproliferation observed in vivo was an indirect effect that was most likely a consequence of the inflammation. These results reveal that loss of SRF disrupts epidermal homeostasis and strongly suggest its involvement in the pathogenesis of hyperproliferative skin diseases, including psoriasis.

Activin is a growth and differentiation factor that controls development and repair of several tissues and organs. Transgenic mice overexpressing activin in the skin were characterized by strongly enhanced wound healing, but also by excessive scarring. In this study, we explored the consequences of targeted activation of activin in the epidermis and hair follicles by generation of mice lacking the activin antagonist follistatin in keratinocytes. We observed enhanced keratinocyte proliferation in the tail epidermis of these animals. After skin injury, an earlier onset of keratinocyte hyperproliferation at the wound edge was observed in the mutant mice, resulting in an enlarged hyperproliferative epithelium. However, granulation tissue formation and scarring were not affected. These results demonstrate that selective activation of activin in the epidermis enhances reepithelialization without affecting the quality of the healed wound.

There is a major discrepancy between the in vitro and in vivo results regarding the role of beta1 integrins in the maintenance of epidermal stem/progenitor cells. Studies of mice with skin-specific ablation of beta1 integrins suggested that epidermis can form and be maintained in their absence, while in vitro data have shown a fundamental role for these adhesion receptors in stem/progenitor cell expansion and differentiation. To elucidate this discrepancy we generated hypomorphic mice expressing reduced beta1 integrin levels on keratinocytes that developed similar, but less severe defects than mice with beta1-deficient keratinocytes. Surprisingly we found that upon aging these abnormalities attenuated due to a rapid expansion of cells, which escaped or compensated for the down-regulation of beta1 integrin expression. A similar phenomenon was observed in aged mice with a complete, skin-specific ablation of the beta1 integrin gene, where cells that escaped Cre-mediated recombination repopulated the mutant skin in a very short time period. The expansion of beta1 integrin expressing keratinocytes was even further accelerated in situations of increased keratinocyte proliferation such as wound healing. These data demonstrate that expression of beta1 integrins is critically important for the expansion of epidermal progenitor cells to maintain epidermal homeostasis.

Insulin-like growth factor 1 (IGF-1) is an important regulator of growth, survival, and differentiation in many tissues. It is produced in several isoforms that differ in their N-terminal signal peptide and C-terminal extension peptide. The locally acting isoform of IGF-1 (mIGF-1) was previously shown to enhance the regeneration of both muscle and heart. In this study, we tested the therapeutic potential of mIGF-1 in the skin by generating a transgenic mouse model in which mIGF-1 expression is driven by the keratin 14 promoter. IGF-1 levels were unchanged in the sera of hemizygous K14/mIGF-1 transgenic animals whose growth was unaffected. A skin analysis of young animals revealed normal architecture and thickness as well as proper expression of differentiation and proliferation markers. No malignant tumors were formed. Normal homeostasis of the putative stem cell compartment was also maintained. Healing of full-thickness excisional wounds was accelerated because of increased proliferation and migration of keratinocytes, whereas inflammation, granulation tissue formation, and scarring were not obviously affected. In addition, mIGF-1 promoted late hair follicle morphogenesis and cycling. To our knowledge, this is the first work to characterize the simultaneous, stimulatory effect of IGF-1 delivery to keratinocytes on two types of regeneration processes within a single mouse model. Our analysis supports the use of mIGF-1 for skin and hair regeneration and describes a potential cell type-restricted action.

Regulated cell proliferation is a crucial prerequisite for Schwann cells to achieve myelination in development and regeneration. In the present study, we have investigated the function of the cell cycle inhibitors p21 and p16 as potential regulators of Schwann cell proliferation, using p21- or p16-deficient mice. We report that both inhibitors are required for proper withdrawal of Schwann cells from the cell cycle during development and following injury. Postnatal Schwann cells express p21 exclusively in the cytoplasm, first detectable at postnatal day 7. This cytoplasmic p21 expression is necessary for proper Schwann cell proliferation control in the late development of peripheral nerves. After axonal damage, p21 is found in Schwann cell nuclei during the initiation of the proliferation period. This stage is critically regulated by p21, since loss of p21 leads to a strong increase in Schwann cell proliferation. Unexpectedly, p21 levels are upregulated in this phase suggesting that the role of p21 may be more complex than purely inhibitory for the Schwann cell cycle. However, inhibition of Schwann cell proliferation is the overriding crucial function of p21 and p16 in peripheral nerves as revealed by the consequences of loss-of-function in development and after injury. Different mechanisms appear to underlie the inhibitory function, depending on whether p21 is cytoplasmic or nuclear.

Recent evidence suggests that changes in membrane potential influence the proliferation and differentiation of keratinocytes. To further elucidate the role of changes in membrane potential for their biological fate, the electrical behavior of keratinocytes needs to be studied under complex conditions such as multilayered cultures. However, electrophysiological recordings from cells in the various layers of a complex culture would be extremely difficult. Given the high spatial resolution of confocal imaging and the availability of novel voltage-sensitive dyes, we combined these methods in an attempt to develop a viable alternative for recording membrane potentials in more complex tissue systems. As a first step, we used confocal ratiometric imaging of fluorescence resonance energy transfer (FRET)-based voltage-sensitive dyes. We then validated this approach by comparing the optically recorded voltage signals in HaCaT keratinocytes with the electrophysiological signals obtained by whole cell recordings of the same preparation. We demonstrate 1) that optical recordings allow precise multisite measurements of voltage changes evoked by the extracellular signaling molecules ATP and bradykinin and 2) that responsiveness to ATP differs in various layers of cultured keratinocytes.

We used a combination of electrophysiological and cell and molecular biological techniques to study the regulation and functional role of the intermediate conductance Ca2+-activated K+ channel, hIK1, in HaCaT keratinocytes. When we incubated cells with the hIK1 opener, 1-ethyl-2-benzimidazolinone (1-EBIO), to investigate the cellular consequences of prolonged channel activity, an unexpected down-regulation of channels occurred within a few hours. The same effect was produced by the hIK1 openers chlorzoxazone and zoxazolamine and was also observed in a different cell line (C6 glioma cells). After 3 days of treatment with 1-EBIO, mRNA levels of hIK1 were substantially diminished and no channel activity was detected. Down-regulation of hIK1 was accompanied by a loss of mitogenic activity and a strong increase in cell size. After withdrawal of 1-EBIO, hIK1 mRNA and channel activity fully recovered and the cells resumed mitogenic activity. Our data present evidence for a novel feedback mechanism of hIK1 expression that appears to result from the paradoxical action of its pharmacological activator during prolonged application. Because the down-regulation of hIK1 bears immediate significance on the biological fate of keratinocytes, 1-EBIO and related compounds might emerge as potent tools to influence the proliferation of various non-excitable cells endowed with IK channels.