Rock Lab @ UCSF

RESEARCH

Rock 2

Section of asthmatic human airway stained with antibodies against cytoskeletal proteins (green) and the basal cell-specific transcription factor p63 (red).

Hundreds of millions of alveoli in the distal lung mediate its vital gas exchange function. This necessitates a thin epithelial lining across which gases can diffuse in close apposition to a dense capillary network. These cells provide an epithelial barrier to insulate our bodies from the outside world. More proximally, a network of airways carries gases to and from the alveoli. More than simple conduits, these airways filter the more than 11,000 liters of gases inhaled by the average human each day. Inhaled microorganisms and particulates are trapped in the mucus that lines the airways and cleared by mucociliary transport. This requires an epithelial lining made up of cells with the appropriate complement of ion channels to maintain airway hydration and the proper balance of secretory cells and multi-ciliated cells for the effective anterograde transport of mucus and foreign material.

We are interested in how environmental pressures and genetic changes perturb homeostasis and contribute to pathological changes that impair lung function. By understanding these effects, we hope to identify genetic and molecular therapies for lung disease. We are also interested in identifying cells with the capacity for long-term self-renewal and multilineage differentiation; these cells are the ideal starting material for cell-based therapies for lung disease or bioengineered organs.

1. IDENTIFICATION OF STEM CELL POPULATIONS IN THE MOUSE AND HUMAN LUNG

Schematic

Proximal airways of mouse and human lungs are lined by a pseudostratified epithelium. We have shown that TRP63+ basal cells of these airways function as a population of stem cells capable of long-term self-renewal and the generation of differentiated daughters (multi-ciliated and secretory cells). Using a combination of in vivo mouse models and in vitro studies with human cells, we showed that the evolutionarily conserved Notch pathway is required for the differentiation of basal stem cells, particularly along secretory (mucous cell) lineages, but is not required for their self-renewal. Bronchioles (more distal airways with smaller diameters) in mice are lined by a simple columnar epithelium made up of multi-ciliated cells and secretory cells (but lack basal cells). Here, evidence suggests that secretory cells (that express Scgb1a1) are stem cells capable of long-term self-renewal and differentiation. Importantly, we currently do not know whether a similar "zone" exists in the human lung; most human airways, right down to the broncho-alveolar duct junction (BADJ), are lined by a pseudostratified epithelium that contains TRP63+ basal stem cells. The alveoli are made up of Type I and Type 2 alveolar epithelial cells (AEC1 and AEC2, respectively). For decades, AEC2 have been regarded as the alveolar epithelial stem cell, but there were no rigorous in vivo genetic lineage tracing data to support this claim. Recently, we showed that AEC2 do give rise to AEC1 under steady state conditions and that the kinetics of this process are enhanced in response to bleomycin-induced lung injury. Importantly, our data (and data from the laboratories of our collaborators Hal Chapman, UCSF and Brigid Hogan, Duke University) support a model in which there is another, non-AEC2 alveolar epithelial stem cell. We are currently using the technique of genetic lineage tracing in mice to test the progenitor potential of putative stem cell populations in vivo during lung development, in adults under steady state conditions, and in response to a variety of lung injuries. We combine gene expression analysis with in vivo and in vitro gain- and loss-of-function experiments to test hypotheses about the mechanisms that regulate the self-renewal and differentiation of stem cells from mouse and human lungs. Our goal is to translate this information into genetic, molecular and cell-based therapies for lung disease.

2. CELLULAR ORIGINS OF PULMONARY FIBROSIS

Fibrosis

Section of a lung from a patient with idiopathic pulmonary fibrosis (IPF). Fibroblasts and extracellular matrix (ECM, red) accumulate in the interstitial space around the airways, alveoli and vasculature (green). These severely limit lung function. By understanding the normal cell lineage relationships in the distal lung and their molecular regulation, we hope to identify novel strategies to prevent or reverse remodeling in pulmonary fibrosis.

Pulmonary fibrosis is a progressive and debilitating lung disease in which the alveolar gas exchange region of the lung is replaced by scar tissue. At least three populations have been proposed as the source of mesenchymal cells (i.e., myofibroblasts) that produce the characteristic fibrotic lesions: (1) epithelial cells, through the process of epithelial-to-mesenchymal transition, (2) circulating fibrocytes and (3) resident stromal cells, including fibroblasts, pericytes and "contractile interstitial cells." We recently combined genetic lineage tracing in the mouse model of bleomycin-induced pulmonary fibrosis with high-resolution confocal microscopic analysis of healthy and fibrotic human lungs to investigate the cellular origins of pulmonary fibrosis. Our data suggest that AEC2 cells and multiple stromal cell types, including CSPG4/NG2+ pericytes and PDGFRA+ "fibroblasts," proliferate in fibrotic lungs. However, neither AEC2 nor pericytes are a source of myofibroblasts. We are interested in identifying subsets of resident stromal populations in healthy lungs and understanding how they contribute, directly or indirectly, to the progression of lung disease.

3. FUNCTIONS OF THE CALCIUM-ACTIVATED CHLORIDE CHANNEL TMEM16A

16aGFP

Section of a trachea from a TMEM16A::GFP fusion protein "knock-in" mouse strain stained with antibodies against GFP (green, functional TMEM16A::GFP fusion protein) and mucin (MUC5AC, red). The abundance of apical abundance of apically localized TMEM16A::GFP (green) is increased, predominantly in secretory cells, in the context of allergic airway disease.

Tmem16a

Section of a gastrointestinal stromal tumor from a mouse model harboring a mutation in the c-kit receptor. Tumor cells were stained with an antibody against TMEM16A (green) and smooth muscle cells with an antibody against aSMA (red).

We have shown that TMEM16A encodes a calcium-activated chloride channel (CaCC) in neonatal mouse airways. Impaired activity of this channel in mouse or human airway epithelial cells causes defects in the secretion of ions and mucus. Because TMEM16A is expressed in human airways (including patients with mutations in another chloride channel, CFTR), pharmacological modulation of this "alternative" chloride channel is regarded as a promising therapeutic strategy for cystic fibrosis. We use mouse models and in vitro assays to test hypotheses about the functions of this channel in a variety of contexts including embryonic development, adult airway physiology, and tumor progression.