Fibroblastos

Evidence that fibroblasts derive from epithelium during tissue fibrosis

Masayuki Iwano1, David Plieth1, Theodore M. Danoff2,3, Chengsen Xue1,Hirokazu Okada3,4 and Eric G. Neilson1,3

1Department of Medicine, and Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee, USA

2 GlaxoSmithKline, Philadelphia, Pennsylvania, USA

3 Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

4 Department of Nephrology, Saitama Medical College, Irumagun, Japan

Address correspondence to: Eric G. Neilson, Department of Medicine, D-3100 Medical Center North, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2358, USA. Phone: (615) 322-3146; Fax: (615) 343-9391; E-mail: Eric.Neilson@Vanderbilt.edu.

Published August 1, 2002

Received for publication March 25, 2002, and accepted in revised form June 11, 2002.

Interstitial fibroblasts are principal effector cells of organ fibrosis in kidneys, lungs, and liver. While some view fibroblasts in adult tissues as nothing more than primitive mesenchymal cells surviving embryologic development, they differ from mesenchymal cells in their unique expression of fibroblast-specific protein-1 (FSP1). This difference raises questions about their origin. Using bone marrow chimeras and transgenic reporter mice, we show here that interstitial kidney fibroblasts derive from two sources. A small number of FSP1+, CD34– fibroblasts migrate to normal interstitial spaces from bone marrow. More surprisingly, however, FSP1+ fibroblasts also arise in large numbers by local epithelial-mesenchymal transition (EMT) during renal fibrogenesis. Both populations of fibroblasts express collagen type I and expand by cell division during tissue fibrosis. Our findings suggest that a substantial number of organ fibroblasts appear through a novel reversal in the direction of epithelial cell fate. As a general mechanism, this change in fate highlights the potential plasticity of differentiated cells in adult tissues under pathologic conditions.

See the related Commentary beginning on page 305.

Introduction

Cell fate pathways for epithelial tissues have overlapping complexities on many levels (1). Pathway integration ultimately determines the migration and interaction of progenitor cells under the control of genetic and morphogenic cues, the timed partitioning of cellular determinants, and plasticity among lineages until terminal differentiation shapes final structure and function (2, 3). With evolving tissue maturity, epithelial units organize as repeating structures, and fibroblasts come to reside in the interstitial spaces that form between functional units. Unfortunately, the order and assembly of these patterned events are not well understood (4, 5); for that matter, not all cells have been studied. The origin of interstitial fibroblasts, for example, has been largely overlooked, and their lineage is inconclusive (6). We undertook the present study because recent availability of new fibroblast markers has reduced the difficulty in addressing this issue (7, 8).

Two hypotheses emerge regarding the origin of adult fibroblasts. One hypothesis argues that marrow stromal cells (MSCs) are progenitors for tissue fibroblasts that then shuttle through the circulation to populate peripheral organs (6, 9–11). While MSCs can migrate to remote tissues and clearly develop a fibroblastic phenotype in culture (6), no evidence exists to show they engage in tissue fibrosis after migration. In fact, most of the recent interest in MSCs focuses on their capacity to give rise to more differentiated cells in nonhematogenous organs (5, 12, 13). A second hypothesis favors epithelial-mesenchymal transition (EMT) in the local formation of interstitial fibroblasts from organ epithelium (7). While many neoteric cell lineages migrate during embryogenesis to new locations using a fate pathway that involves EMT (14, 15), such transitions in mature tissues are less well appreciated. However, transitions do occur among adult cells (5), particularly during oncogenesis (16) and fibrotic tissue repair following injury — a process known as fibrogenesis (7, 17).

The appeal of an argument for EMT in the formation of fibroblasts is its simplicity; fibroblast dispersal in local interstitial spaces is assured by local epithelium, particularly when fibroblasts are needed for fibrogenesis. Indirect support for this notion stems from earlier work that identified fibroblast-specific protein-1 (FSP1) as an EMT marker in cultured epithelial cells undergoing transition to fibroblasts (18), as well as histologic evidence in vivo that epithelial units expressing FSP1 disaggregate as organ tissues dedifferentiate during the early stages of fibrogenesis (7, 19).

Epithelial cells sit on and attach to basement membranes that provide context and architectural stability for the cell-cell contact emblematic of this phenotype. When basement membrane is damaged by proteases or disrupted by alterations in assembly, epithelia begin to express cytokines that initiate EMT (20). Growth factors such as TGF-β, EGF, and FGF-2 facilitate EMT by binding epithelial receptors with ligand-inducible intrinsic kinase activity (16, 21, 22). The activation of Ras and Src pathways (16) and a shift in the balance of small GTPase activity (23) provide important transcriptional signals for loss of adhesion (24) and induction of EMT in cultured cells. In performing these functions, TGF-β and EGF also induce the expression of FSP1 in transitioning tubular epithelium (18). FSP1 is a fibroblast-specific protein in the S100 class of cytoplasmic, calcium-binding proteins (7). The members of this family have been implicated in microtubule dynamics, cytoskeletal membrane interactions, calcium signal transduction, p53-mediated cell cycle regulation, and cellular growth and differentiation. While the precise function of FSP1 and its homologues is not entirely clear, its interaction with non-muscle myosin II, tropomyosin, actin, or tubulin, and the inducibility of rearrangements in F-actin stress fibers suggest that FSP1 may be associated with mesenchymal cell shape and motility. Inhibition of EMT in cultured cells by blocking FSP1 expression implies a direct role in reshaping cytoskeletal architecture (18).

While EMT is well described in cultured cells, it remains to be shown whether such events occur in adult vertebrate tissues. Furthermore, although fibroblasts readily proliferate in culture when bombarded with cytokines and serum, they are generally quiescent in normal tissue. In preliminary in vivo experiments in normal mice expressing a transgene in tissue fibroblasts that encodes for thymidine kinase under the control of the FSP1 promoter (25), FSP1+ fibroblasts were not reduced in number by DNA chain termination until they randomly entered the cell

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