Analysis of ILK function in vivo/characterization of keratinocyte-specific ILK

3.3. Analysis of ILK function in vivo/characterization of

at an age of 4w. Deletion of ILK leads to progressive hair loss and reticular pigmentation of the skin.

Immunostaining was done by Michal Grzejszczyk and Katrin Lorenz. Pictures in C. were taken by Dr. Takao Sakai.

At birth ILK-K5 mice were indistinguishable from control littermates. After 1-2 weeks when control animals developed a dense hair coat, ILK-K5 mice displayed only scattered hair with partial alopecia. By 4 weeks of age ILK-K5 mice had lost almost all hair and displayed reticular skin pigmentation (Fig 3.14C). However, ILK-K5 mice were of normal size and had a normal life span.

3.3.2. ILK-K5 mice display severe epidermal and HF abnormalities

The epidermis of ILK-K5 mice was normal until P2 but became progressively hyperplastic (Fig 3.15A). While basal keratinocytes were polarized and firmly attached to the BM in control mice they appeared flattened in ILK-K5 mice and frequently detached from the underlying BM (Fig 3.15B). This skin blistering became more severe with age.

In addition to these epidermal defects, loss of ILK severely impaired the development of HFs (Fig 3.15A, C), which diverged into two subpopulations in ILK-K5 mice. At P14 around 66%

of the HFs were arrested in their development and showed no hair shaft formation, a misshapen hair matrix and DP (Fig 3.15A, C; ■). About 33% of the HFs were able to complete HF morphogenesis, but were shorter and characterized by a substantial hyperplasia of the ORS (Fig 3.15A, C; ▲). A plausible explanation for the two HF populations is likely the combination of asynchronous HF morphogenesis and the perinatal deletion of the ILK protein. While fully developed HFs might have lost ILK late during morphogenesis, early arrested HFs lost ILK most likely at earlier developmental stages.

No further hair cycle was induced in ILK-K5 mice as demonstrated by histological analysis of back skin section from P28-old mice. By 10w of age all HFs of ILK-K5 mice were resorbed and the melanin deposits gave rise to the reticular skin pigmentation. These data show that ILK is essential for epidermal integrity and HF morphogenesis.

Fig 3.15. Histological analysis of control and ILK-K5 back skin sections. A. Hematoxylin/Eosin staining of back skin sections from control and ILK-K5 mice. Loss of ILK leads to skin blistering and the development of two HF populations (■ arrested HFs, ▲ developed HFs). At later stages HFs are completely lost in ILK-K5 mice. D: dermis, E: epidermis, PC: panniculus carnosum. Scale bar: 100µm. B. Hematoxylin/Eosin staining of back skin sections from control and ILK-K5 mice at P14. Loss of ILK leads to epidermal detachment (*) from the underlying dermis. C. High magnification of HFs from control and ILK-K5 back skin sections at P9. Developed HFs (▲) display a multilayered ORS (*). Scale bar: 50µm. Histology and immunostaining was performed by Michal Grzejszczyk and Katrin Lorenz.

3.3.3. Loss of ILK impairs integrin expression and BM integrity

The detachment of ILK-K5 epidermis from the underlying dermis (Fig 3.15B) indicated an impaired integrin-BM interaction in ILK-deficient keratinocytes. To address this point, back skin sections from 2w-old mice were analyzed by immunostaining for β1 and β4 integrins as well as the BM marker laminin332. While in control mice β1 and β4 integrins were expressed exclusively in basal keratinocytes and enriched along the dermal-epidermal junction, these integrin subunits were localized basally but also frequently found on suprabasal keratinocytes (Fig 3.16A) in ILK knockout mice. In addition the BM was severely distorted in ILK-K5 mice. While control skin displayed a linear laminin332 staining along the dermal-epidermal junction, ILK-K5 skin showed an irregular laminin332 staining and areas of massive laminin diffusion into the dermis (Fig 3.16B). These BM defects were confirmed by ultrastructural analyses (in collaboration with Dr. Wilhelm Bloch, University of Cologne) which, however, also revealed that hemidesmosomes could form in ILK-K5 epidermis (Fig 3.16C). Double immunostaining for the BM marker nidogen and phalloidin revealed that in control epidermis actin is restricted to the apical and lateral plasma membrane, whereas in ILK-K5 mice f-actin was also present at the basal side facing the BM (Fig 3.16D).

Therefore it can be concluded that loss of ILK is essential for the integrity of the epidermal BM as well as the polarization of the f-actin cytoskeleton in basal keratinocytes.

3.3.4. ILK is not required for keratinocyte proliferation

β1 integrin expression is thought to determine the proliferation potential of keratinocytes (Carroll et al. 1995; Jones et al. 1995) and deletion of β1 integrins in basal keratinocytes indeed diminishes keratinocyte proliferation in vivo (Brakebusch et al. 2000). To test whether ILK is essential for the proliferation of keratinocytes, histological sections were analyzed by Ki67 immunostaining. While no significant differences in the proliferation of basal keratinocytes were observed, we detected a significant number of proliferating cells in suprabasal cell layers (Fig 3.16E). Double immunostaining of integrins and proliferation markers revealed that these suprabasal cells were those cells which still expressed β1 and β4 integrins (Fig 3.16A, B). These data indicate that the ectopic location of basal keratinocytes most likely caused by an impaired adhesion to the BM contributes to the epidermal thickening. More importantly, these data show that ILK is not required for β1 integrins to induce proliferation.

Fig 3.16. Suprabasal integrin expression and impaired BM integrity in the absence of ILK. A. Immunostaining of back skin sections from 2w-old mice. β1 integrin was frequently localized on suprabasal keratinocytes (*).

Laminin332 staining was irregular and indicated massive diffusion of laminin into the dermis. B. β4 integrin was frequently localized around suprabasal cells. C. Electron micrographs of back skin section from 2w-old mice showing the impaired BM structure in ILK-K5 skin. Hemidesmosomes can form in the absence of ILK (arrowhead) D. Immunostaining of back skin sections from 2w-old mice immunostained for nidogen and f-actin.

F-actin was mislocalized to basal sides in ILK-K5 epidermis (see arrowheads). E. Proliferating suprabasal cells were frequently detected in the hyperthickened epidermis of ILK-K5 mice (*). Immunostaining for A, B and D were done by Michal Grzejszczyk and Katrin Lorenz. Electron microscopy was performed by Dr. Wilhelm Bloch (University of Cologne). Scale bar: 25µm.

3.3.5. Accumulation of proliferating cells in the ORS of ILK-deficient HFs

Deletion of β1 integrin in the epidermis leads to reduced proliferation of epidermal keratinocytes but also hair matrix cells (Brakebusch et al. 2000). To investigate whether altered proliferation is the reason for impaired HF morphogenesis, back skin section of

mice were subjected to BrdU incorporation assays. KI67 immunostaining revealed the presence of an increased number of proliferating cells in the ORS of both developed and growth arrested mutant HFs (Fig 3.17A). Quantification of BrdU-positive cells in P7 and P14 HFs confirmed this observation. While at P7 the number of proliferating ORS cells was only slightly increased in mutant HFs, this difference became more obvious at P14 (Fig 3.17B).

Interestingly, at the same time the number of BrdU-positive cells was decreasing in the hair matrix (HM) of ILK-K5 HFs (Fig 3.17C) leading to a reduced total number of HM cells (Fig 3.17D). These data suggested that defective morphogenesis of ILK-K5 HFs is not caused by reduced proliferation but rather by an impaired downward migration of ORS cells to the hair matrix.

Fig 3.17. ILK-deficient HFs accumulate proliferating cells in the ORS. A. Ki67 immunostaining of control and mutant HFs at P7. Note the accumulation of proliferating cells along the ORS of mutant HFs (*). Scale bar:

50µm. B. Increased number of positive cells in the ORS of ILK-K5 HFs. C. Reduced number of BrdU-positive cells in the HM of ILK-K5 HFs. D. The total number of cells in the HM is reduced in ILK-K5 HFs.

Immunostaining was done by Michal Grzejszczyk and Katrin Lorenz.

3.3.6. ILK is essential for directional cell migration

To test the hypothesis that loss of ILK impairs the migration of ORS cells along the BM that lines the HF, primary keratinocytes were isolated from control and ILK-K5 mice and analyzed in vitro. Time-lapse microscopy revealed that ILK-deficient keratinocytes are not able to migrate in a persistent manner. Control cells usually formed broad and stable lamellipodia, which allowed single cells to directionally migrate. In contrast, ILK-deficient keratinocytes formed highly unstable lamellipodia which were frequently collapsing. New lamellipodia formed at different locations simultaneously leading to frequent changes of migration direction and hence prohibited a persistent cell migration (Fig 3.18).

Fig 3.18. ILK is essential for directional migration of keratinocytes. Keratinocytes from control and mutant mice were seeded on a FN/ColI matrix and analyzed by time-lapse microscopy. Control cells were able to perform directional migration, while ILK-K5 cells frequently changed their direction due to highly instable lamellipodia. (Red arrows indicate the retracting area of the cells. Green arrows indicate areas of protrusive activity). Scale bars: 10µm. Isolation of keratinocytes was done by Katrin Lorenz.

3.3.7. Loss of ILK is essential for stress fiber formation and establishment of mature FAs in keratinocytes

To test, if the reduced stability of lamellipodia is caused by impaired FC and FA formation which impairs the fixation of this structure to the substrate, primary keratinocytes were analyzed by immunostainings. Visualization of the f-actin cytoskeleton by phalloidin staining and of FAs by FAK immunostaining revealed that ILK-deficient keratinocytes were not able to assemble strong bundles of f-actin (Fig 3.19A). Moreover, the formation of FAs was

mutant keratinocytes revealed a reduced activation of FAK in the absence of ILK (Fig 3.19B) which is in line with the reduced number of FAs in ILK-K5 keratinocytes. To check whether reduced activation of Rac1 contributes to the migration defect of ILK-K5 keratinocytes (Fig 3.18), primary cells were subjected to Rac1 pulldown assays during cell adhesion (Fig 3.19C) or after growth factor stimulation (Fig 3.19D). Interestingly, no significant differences in Rac1 activation levels could be detected.

These data indicate that the reduced migration of ILK-deficient keratinocytes is mainly caused by impaired cell adhesion due to defective FA formation and maturation as well as a disturbed formation of the f-actin cytoskeleton.

Fig 3.19. Loss of ILK leads to impaired stress fiber formation and FA assembly in keratinocytes. A.

Immunostaining of control and ILK-K5 primary keratinocytes revealed that loss of ILK impaired FA and stress fiber formation. Moreover most of the ILK-K5 keratinocytes were much smaller. Scale bar: 10µm in ILK Co and 20µm in ILK-K5. B. Biochemical analysis of protein lysates from primary keratinocytes showed reduced activation levels of FAK in ILK-K5 keratinocytes. C. Western blot analysis of Rac1 pulldown assays revealed no significant differences in Rac1 activation in control and ILK-K5 primary keratinocytes in suspension (0) or 30min after cell adhesion to a laminin-rich matrix. D. Western blot analysis of a Rac1 pulldown assay showing no significant differences after growth factor-induced Rac1 activation in control and ILK-K5 primary keratinocytes. Cells were stimulated for 15min and 30min with 8% FCS. Immunostaining in A was done by Katrin Lorenz and Dr. Robert Torka.

3.4. Analysis of ILK function in vitro/Characterization of ILK knockout