Regulation of Keratin Expression During Differentiation and Development 
in Human Skin 
tal architecture of keratin filaments may be specif- 
ically tailored to suit the particular structural 
needs of each epidermal cell at various stages of 
differentiation and development. 
To determine the molecular details of the fila- 
ment assembly process and to investigate the na- 
ture of interactions of keratin filaments with 
other proteins and organelles, we have used dele- 
tion and site-directed mutagenesis to engineer al- 
terations in the coding sequences of K5 and Kl4, 
the keratin pair expressed in the living cells of 
the epidermis. To generate substantial quantities 
of keratins for filament assembly studies, we used 
genetic engineering to overexpress wild-type and 
mutant human keratins in bacteria, which do not 
have keratin filament networks. We purified 
these keratins and examined the consequences of 
mutations and deletions on keratin filament as- 
sembly in vitro. We have begun to identify those 
sequences involved in filament elongation and 
those that are more important for lateral associa- 
tions. In addition, we are using gene transfection 
of human tissue culture cells to examine 1) the 
dynamics of keratin filament assembly in vivo 
and 2) how the abnormal expression of keratins 
in malignant and hyperproliferating epidermal 
cells might change their biology and cytoskeletal 
architecture. The results of these studies have be- 
gun to yield valuable insights into the complex 
assembly process of keratin filaments and into the 
function that these filaments perform in provid- 
ing a relatively indestructible skin surface. 
In addition to examining the structure of kera- 
tin filaments, we have also been interested in de- 
termining whether there might be natural muta- 
tions in human epidermal keratin genes that 
could lead to a genetic skin disease. We had previ- 
ously shown that elevated epidermal keratin gene 
expression is a relatively late event in develop- 
ment and that certain keratin mutants have a dom- 
inant effect in cultured cells (i.e., they integrate 
into and disrupt the endogenous keratin 
filament network). In the past year, we made 
transgenic mice and used epidermal keratin pro- 
moters to target expression of some of these domi- 
nant mutant keratins to the mouse epidermis. Un- 
expectedly, the transgenic mice exhibited a 
phenotype that strongly resembles that of pa- 
tients with epidermolysis bullosa simplex (EBS), 
a dominant and sometimes devastating, blistering 
human skin disease of unknown etiology. We are 
currently examining patients with EBS to deter- 
mine whether they have mutations in their epi- 
dermal keratin genes. 
We are also analyzing the complexity of the 
multiple keratin genes in the human genome and 
the mechanisms underlying their differential reg- 
ulation. We have already shown that keratin ex- 
pression is controlled at the transcriptional level 
and that it is regulated by vitamin A. The two 
genes expressed in the basal layer of the epider- 
mis have been isolated and characterized, and we 
are focusing on how these genes are regulated 
during development and how their expression is 
influenced by retinoids. In the past year, we iden- 
tified proximal and distal domains that synergisti- 
cally act to regulate expression of one of these 
basal genes. Our goal is to identify the sequences 
and transcription factors involved in the regula- 
tion of these genes. This analysis of the factors 
controlling the cell's major structural genes 
should lead us to the factors involved in deter- 
mining keratinocyte fate. Unraveling the nature 
of promoter and enhancer sequences involved in 
regulating epidermal genes will not only be im- 
portant for understanding epidermal develop- 
ment but may also be useful for targeting prod- 
ucts to the epidermis, e.g., in drug therapy. 
Because epidermal cells can be removed from a 
patient, cultured in vitro, and grafted back onto 
the patient, such techniques are potentially use- 
ful for medical research. 
Knowledge of the normal regulation processes 
of epidermal and hair differentiation will be es- 
sential to identify the points at which these pro- 
cesses go awry in different genetic skin diseases 
and skin cancers. Elucidating the molecular na- 
ture of the normal and abnormal programs of dif- 
ferentiation in the skin should lead to new and 
badly needed treatments for dermatological 
diseases. 
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