MOLECULAR BIOLOGY OF THE SKIN 
Elaine Fuchs, Ph.D., Investigator 
The global objective of this laboratory is to under- 
stand the molecular mechanisms underlying epider- 
mal differentiation and development. Epidermal 
cells manifest their protective function by building 
an extensive cytoskeletal architecture of 10-nm ker- 
atin filaments (intermediate filaments, IPs), which 
account for 30% of basal cell protein and 85% of 
protein in fully differentiated squamae. As keratino- 
cytes commit to differentiate, they switch off ex- 
pression of K5 and K14, which form dispersed fila- 
ments, and switch on expression of Kl and KIO, 
which assemble into filaments that bundle. This pro- 
gressive increase in filament bundling seems to en- 
able IPs to be among the few survivors of the massive 
destructive phase that ensues as a cell becomes meta- 
bolically inert and reaches the skin surface. A knowl- 
edge of the function and structure of keratin IPs and 
how keratin genes are regulated is prerequisite to 
understanding epidermal differentiation and the 
possible involvement of keratin mutations in ge- 
netic disease. 
Function and Structure of Keratin 
Filaments, and Relevance to Genetic 
Skin Disorders 
Approximately 10,000 coiled-coil heterodimers 
of type I and type II keratins form stable heterote- 
tramers that then self-assemble into each IF. To elu- 
cidate this basic subunit structure and to determine 
the mechanisms underlying assembly, this labora- 
tory conducted deletion and site-directed mutagen- 
esis of cDNAs encoding human K5 and Kl4. Por in 
vitro filament assembly studies, mutant and wild- 
type keratin cDNAs were overexpressed in bacteria, 
and biochemical methods were used to purify milli- 
gram quantities of human keratins. Biochemical and 
electron microscopic studies revealed that 1) the 
nonhelical tail domains play a role in filament stabi- 
lization, 2) the nonhelical head domain of K5 and 
the central a-helical rod segments of K5 and K 1 4 are 
essential for IP structure, 3) rod-deletion mutants 
interfere predominantly with end-to-end rather than 
with lateral interactions, and 4) even subtle point 
mutants in the highly conserved rod ends are more 
deleterious to IP assembly than are proline muta- 
tions within the rod. These studies were the first to 
use molecular genetics to examine IP assembly in 
detail. 
Dr. Puchs and her colleagues pioneered methods 
to examine effects of IP deletions on cytoskeletal 
networks of cultured cells. They then prepared 
transgenic mice expressing severe and mild IP- 
disrupting mutants of the human Kl 4 gene. Surpris- 
ingly, these mice exhibited a striking resemblance 
to a group of human blistering skin diseases known 
as epidermolysis bullosa simplex (PBS). Upon mild 
physical trauma, their skin blistered because of cy- 
tolysis of basal epidermal cells. Mice expressing se- 
verely disrupting Kl4 mutants resembled the most 
severe form, Dowling-Meara EBS, with large clumps 
of keratin in the cytoplasm of their basal cells. Mice 
expressing milder Kl4 mutants resembled milder 
forms of EBS, with disorganization but no gross 
clumping of keratin protein. These results suggest 
strongly that 1 ) EBS arises from defects in the forma- 
tion of K5/K14 filament networks, and 2) the multi- 
ple forms of EBS are genetically related, arising from 
different defects in the K14/K5 genes. Pinally, the 
correlation between perturbation of keratin IPs and 
cell cytolysis on mechanical stress indicates that an 
important function of keratin IPs is to impart me- 
chanical integrity to epidermal cells. 
A major focus of last year was to isolate and charac- 
terize the Kl4 and K5 cDNAs/genes from patients 
with EBS. In two distinct incidences of Dowling- 
Meara EBS, point mutations were found in the Kl4 
gene, in a codon for a highly conserved arginine 
residue at the amino terminus of the Kl4 rod do- 
main. Using genetic engineering, gene transfection, 
and in vitro filament assembly. Dr. Puchs and her 
colleagues showed that these mutations are func- 
tionally responsible for the basal cell tonofilament 
clumping that is a hallmark of the disease. 
This research on EBS suggests that a second ge- 
netic skin disease, epidermolytic hyperkeratosis 
(EH), might be a disorder of the differentiation- 
specific keratins Kl and KIO. EH is characterized by 
tonofilament clumping and cytolysis in suprabasal 
rather than in basal cells. To address this possibility, 
a truncated version of the human KIO gene was gen- 
erated. In mice this mutant caused the clinical and 
biochemical manifestations of EH. These findings 
prompted an investigation of human EH, and re- 
cently Dr. Puchs and her colleagues discovered a 
point mutation in the human KIO gene of two unre- 
lated families with EH. The mutation is present only 
in affected family members, and subsequent analysis 
demonstrated that it is functionally responsible for 
the clinical manifestations of EH in these patients. 
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