Myofibroblasts are the major cellular source of collagen, and their accumulationvia differentiation from fibroblasts and resistance to apoptosis-is a hallmark of tissue fibrosis. Clearance of myofibroblasts by de-differentiation and restoration of apoptosis sensitivity has the potential to reverse fibrosis. Prostaglandin E2 (PGE2) and mitogens such as FGF2 have each been shown to de-differentiate myofibroblasts, but the resultant cellular phenotypes have neither been comprehensively characterized nor compared. Here we show that PGE2 elicited de-differentiation of human lung myofibroblasts via cAMP/PKA while FGF2 utilized MEK/ERK. The two mediators yielded transitional cells with distinct transcriptomes, with FGF2 promoting but PGE2 inhibiting proliferation and survival. The gene expression pattern in fibroblasts isolated from the lungs of mice undergoing resolution of experimental fibrosis resembled that of myofibroblasts treated with PGE2 in vitro. We conclude that myofibroblast dedifferentiation can proceed via distinct programs exemplified by treatment with PGE2 and FGF2, with that occurring in vivo most closely resembling the former.

The base excision repair (BER) pathway recognizes and repairs most non-bulky lesions, uracil and abasic (AP) sites in DNA. Several participants are embryonic lethals in knockout mice. Since the pathway has never been investigated during embryogenesis, we characterized the first three steps of BER in zebrafish extracts from unfertilized eggs, embryos at different developmental stages and adults. Using a 45-mer double stranded substrate with a U/G mispair at position 21, we showed that extracts from all stages are capable of performing BER. Before 3 dpf aphidicolin-sensitive polymerases perform most nucleotide insertion. In fact, eggs and early stage embryos lack DNA polymerase-β protein. After hatching at 3 dpf, an aphidicolin-resistant polymerase, probably DNA polymerase β, becomes the primary polymerase. Previously we showed that when zebrafish AP endonuclease protein (ZAP1) level is knocked down, embryos cease dividing after the initial phase of rapid proliferation and die without apoptosis shortly thereafter. Nevertheless, extracts from embryos in which ZAP1 has been largely depleted process substrate equally as well as extracts from control embryos. Since apex1 and apex2 are both strongly expressed in early embryos relative to adults, these data indicate that both may play important roles in DNA repair in early development. In brief, the major differences in BER performed by early stage embryos and adults are the absence of DNA polymerase-β, leading to predominance of replicative polymerases, and the presence of backup Mg2+-dependent endonuclease activity in early stage embryos. The switch to normal, adult BER occurs fully when the embryos hatch from the chorionic membrane and encounter normal oxidative stress.

There is a paucity of information about potential molecular brakes on the activation of fibroblasts that drive tissue fibrosis. The transcription factor Kruppel-like factor 4 (KLF4) is best known as a determinant of cell stemness and a tumor suppressor. We found that its expression was diminished in fibroblasts from fibrotic lung. Gain-and loss-of-function studies showed that KLF4 inhibits fibroblast proliferation, collagen synthesis, and differentiation to myofibroblasts, while restoring their sensitivity to apoptosis. Conditional deletion of KLF4 from fibroblasts potentiated the peak degree of pulmonary fibrosis and abrogated the subsequent spontaneous resolution that follows in a model of transient fibrosis. A small molecule inducer of KLF4 was able to restore its expression in fibrotic fibroblasts and elicit resolution in an experimental model characterized by more clinically relevant persistent pulmonary fibrosis. These data identify KLF4 as a pivotal brake on fibroblast activation whose induction represents a new therapeutic approach in fibrosis of the lung, and perhaps other organs.

PGE 2 and PGI 2 receptors are potential targets for the treatment of chronic lung disease.

The base excision repair (BER) pathway recognizes and repairs most non‐bulky lesions, uracil and abasic (AP) sites in DNA. Since the pathway has never been investigated during embryogenesis, we characterized BER in zebrafish extracts from unfertilized eggs, embryos at different developmental stages and adults. Using a 45‐mer double stranded substrate with a U/G mispair at position 21, we showed that all extracts are capable of performing BER. Before 3 dpf aphidicolin‐sensitive polymerases perform most nucleotide insertion. After hatching, an aphidicolin‐resistant polymerase, probably DNA polymeraseß, becomes the primary polymerase. Previously we showed that when zebrafish AP endonuclease protein (ZAP1) level is knocked down, embryos cease dividing after the initial phase of rapid proliferation and die without apoptosis shortly thereafter. Nevertheless, extracts from embryos in which ZAP1 has been largely depleted process substrate equally as well as extracts from control embryos. Since apex1 and apex2 are both strongly expressed in early embryos relative to adults, these data indicate that both may play important roles in DNA repair in early development. In brief, the major differences in BER performed by early embryos and adults are the predominance of replicative polymerases and the presence of backup Mg2+‐dependent endonuclease activity in early stage embryos. Supported by funds from the National Institutes of Health 1R03HD045517, the G. Harold and Leila Y. Mathers Foundation, Aid for Cancer Research, and Northeastern University