Smoking-induced EGF-dependent Reprogramming of Airway Basal Cell Function

Airway epithelium is composed of 4 major cell types, including ciliated cells, secretory
cells, undifferentiated columnar cells, and basal cells (BC). The earliest changes associated
with the development of smoking-induced lung diseases, such as chronic obstructive pulmonary
disease (COPD) and lung cancer, occur in the airway epithelium, including BC hyperplasia,
squamous metaplasia, mucous cell hyperplasia and metaplasia, impaired ciliated cell structure
and function, loss of Clara cells, and increased epithelial permeability due to impaired
junctional barrier. We hypothesize that fundamental to these changes are smoking-induced
derangements of BC, the stem/progenitor cell population that can self-renew and differentiate
into ciliated and secretory cells. Using technologies established in our laboratory to
culture pure population of BC from the human airway epithelium, to induce differentiation of
these BC in air-liquid interface, and to assess the transcriptome of purified BC compared to
that of the complete differentiated airway epithelium, our preliminary data indicates that:
(1) airway BC exhibit a distinct gene expression signature relevant to stem/progenitor cell
function, including high expression of the epidermal growth factor receptor (EGFR); (2)
airway BC from healthy smokers have a different gene expression pattern compared to
nonsmokers, with enrichment of functional categories related to cell cycle and proliferation
and down-regulation of differentiation-associated genes; and (3) constitutive EGF expression
in BC and differentiated cells is barely detectable, but smoking selectively up-regulates EGF
expression in differentiated cells of the airway epithelium in vivo. Based on these data and
on the knowledge that EGFR signaling plays a central role in the regulation of cell
proliferation and differentiation in the airway epithelium, the central concept of this
proposal is that smoking-induced expression by differentiated cells activates BC via EGFR
altering the molecular phenotype of airway BC and impairing their ability to generate normal
differentiated airway epithelium. To assess this concept, the following aims will be
addressed:

Aim 1. To determine whether stimulation of airway BC from healthy nonsmokers with EGF induces
genes and pathways related to smoking-associated phenotypes, e.g. BC hyperplasia, squamous
metaplasia, mucous metaplasia, abnormal cilia, decreased Clara cell number and compromised
junctional integrity.

Aim 2. To test the hypothesis that stimulation of airway BC from healthy nonsmokers with EGF
alters BC differentiation in air-liquid interface culture, with generation of
smoking-associated phenotypes (see Aim 1).

Aim 3. To test the hypothesis that upon apical exposure to cigarette smoke extract,
differentiated airway epithelial cells derived from BC of healthy nonsmokers release
increased amounts of EGF into the apical supernatant, which will alter the ability of BC of
healthy nonsmokers to generate normal differentiated airway epithelium, and that blocking EGF
in this supernatant will abolish this effect.

Inclusion Criteria:

- Must be capable of providing informed consent

- Males and females, age 18 or older

- Good overall health without history of chronic lung disease, including asthma, and
without recurrent or recent (within 3 months) acute pulmonary disease

- Normal physical examination

- Normal routine laboratory evaluation, including general hematologic studies, general
serologic/ immunologic studies, general biochemical analyses, and urine analysis

- Negative HIV serology

- Normal chest X-ray (PA and lateral)

- Normal electrocardiogram

- Females - not pregnant

- No history of allergies to medications to be used in the bronchoscopy procedure

- Not taking any medications relevant to lung disease or having an effect on the airway
epithelium Willingness to participate in the study

Exclusion Criteria:

- Unable to meet the inclusion criteria

- Pregnancy

- Current active infection or acute illness of any kind

- Current alcohol or drug abuse

- Evidence of malignancy within the past 5 years