Regulation of Keratin Expression During Differentiation and Development 
in Human Skin 
In the past few years, we identified those se- 
quences involved in filament elongation and 
those that are more important for lateral associa- 
tions. In addition, we used gene transfection of 
human tissue culture cells to examine the dy- 
namics of filament assembly in vivo and to deter- 
mine how abnormal expression of keratins in ma- 
lignant and hyperproliferating epidermal cells 
might change their biology and cytoskeletal ar- 
chitecture. The results of these studies have be- 
gun to yield valuable insights into the complex 
assembly process of keratin filaments and the 
function they perform in providing a tough skin 
surface. 
We have also been interested in determining 
whether natural mutations in human epidermal 
keratin genes might lead to genetic skin diseases. 
We had previously shown that elevated expres- 
sion of epidermal keratin genes is a relatively late 
event in development and that certain keratin 
mutants have a deleterious effect in cultured 
cells, disrupting the endogenous keratin filament 
network. In the past two years, we have made 
transgenic mice and used epidermal keratin pro- 
moters to target expression of some of these domi- 
nant mutant keratins to the epidermis. 
Unexpectedly, the transgenic mice exhibit a 
phenotype resembling that of humans with epi- 
dermolysis bullosa simplex (EBS) , a class of blis- 
tering skin diseases that are dominant, sometimes 
life-threatening, and of previously unknown etiol- 
ogy. Mice expressing Kl4 mutants that severely 
disrupt filament assembly exhibit severe blister- 
ing over body trunk regions — lesions resembling 
those of Dowling Meara EBS, the severest form; 
while mice expressing Kl4 mutants that mildly 
perturb the network exhibit blistering only over 
paws, as in the mildest form of EBS, or Weber- 
Cockayne, where patients show blistering pre- 
dominantly on their hands and feet. Thus we 
were able to demonstrate that multiple mutations 
in a single gene, namely K14, can give rise to 
most if not all forms of EBS, thus strongly suggest- 
ing that these diseases may be genetically linked. 
Further studies enabled us to demonstrate that 
skin blistering in EBS is due to the fragility of 
basal epidermal cells caused by an abnormal ker- 
atin filament network, thereby compromising the 
mechanical strength of this cell layer. 
The similarities between the phenotypes in 
mice and humans prompted us to focus on this 
disease. In the past year we characterized the Kl 4 
and K5 mRNAs, genes and proteins from two pa- 
tients with Dowling Meara EBS. Both patients 
have point mutations in an amino acid of the Kl4 
protein that we had previously shown to be criti- 
cal for filament assembly. When we engineered 
and tested the two mutations in our wild-type 
K14 gene, we verified that these defects are re- 
sponsible for the phenotype of the EBS patients. It 
will take much work to develop better diagnostic 
and therapeutic tools for EBS, but elucidating the 
genetic basis is an important first step. 
While EBS is a disease of defects in the coding 
portions of keratin genes, there are other skin dis- 
eases that appear to arise from abnormalities in 
the control of epidermal genes or genes that influ- 
ence growth and/or differentiation. As a prerequi- 
site to investigating the bases for these types of 
diseases, we are 1 ) analyzing the molecular mech- 
anisms underlying the differential expression of 
epidermal keratin genes and 2) utilizing our Kl4 
promoter to make transgenic mice that overex- 
press various regulatory factors — e.g., transform- 
ing growth factor-a (TGFa), epidermal growth 
factor (EOF), cytokines, hormone receptors, and 
proto-oncogenes in the epidermis. 
In the past few years, we have identified proxi- 
mal and distal domains that act synergistically to 
regulate expression of the human Kl4 gene. Our 
goal is to identify the sequences and transcription 
factors involved. Analysis of the factors control- 
ling the cell's major structural genes should lead 
us to the factors determining keratinocyte fate. 
Unraveling the nature of promoter and enhancer 
sequences involved in regulating epidermal 
genes will be important not only for understand- 
ing epidermal development but also for targeting 
products to the epidermis. In the last year, we 
used the Kl4 promoter/enhancer to engineer 
transgenic mice that overexpress TGFa, the major 
autocrine growth factor of the epidermis. Our re- 
sults have shown that TGFa plays an important 
role in controlling the thickness of the epidermis 
and that certain features of TGFa overexpression 
are similar to those of psoriasis, a hyperprolifera- 
tive skin disease. 
The Kl4 promoter/enhancer, so effective in 
transgenic technology, should also be useful for 
drug therapy. Because epidermal cells can be re- 
moved from a patient, cultured in vitro, and 
grafted back, it should be possible to introduce 
foreign genes, driven by the Kl4 promoter/en- 
hancer, into the cultured epidermal cells prior to 
grafting. Such techniques have potentially power- 
ful applications for future medical research. 
148 
