In this study, we hypothesized that changes in alveolar oxygen tension at birth could serve a physiologic function to signal expression of lung genes required for postnatal lung development. Our microarray data revealed that there is an acute change in the level of expression of 157 genes within 2 hours of birth in room air, and the number of gene changes steadily declines at 6, 12 and 24 hours thereafter. Most of the lung gene changes through 6 hours involved transient induction or repression of expression. And less than 30% of the changes involved genes in common to any two successive time points, suggesting sequential waves of new gene expression over time. Four functional categories of gene expression were overrepresented in the microarray data at 2 hours after birth in room air: transcriptional regulation, structure, apoptosis and antioxidant activity. The presence of antioxidants as a functional gene category correlates with our glutathione disulfide data, which shows the presence of transient oxidant stress in the lung at 2 hours, and suggests a response to a change in the local redox state of the lung as environmental oxygen is altered at birth.
Exposure to 10% oxygen at the end of gestation did not significantly alter baseline gene expression in the fetal lung. Nor did it adversely impact survival of fetal or newborn mice up to 24 hours after birth suggesting that changes in blood flow, lung stretch, nutrient supply and fluid resorption were similar to that of mice birthed in room air and less likely to differentially impact gene expression. Rather birth into 10% oxygen was associated with a 60% decrease in the number of gene changes at 2 hours, compared to that in room air, elimination of any overrepresented functional category of gene expression and attenuation of glutathione disulfide accumulation. Hence birth into an environment of hypoxia is associated with retention of a gene expression pattern like that of the fetal lung.
With birth into room air, there were also fewer gene expression changes in the liver at 2 hours compared to the lung, as well as a lack of overrepresentation of any functional category of gene expression. Interestingly, oxidant stress was not evident in the liver at these early time points after birth as glutathione disulfide did not accumulate. The liver may not be subject to the same magnitude of change in environmental oxygen tension as the lung or it may follow a different time course. The few gene expression levels that did change in common between these two organs could suggest a shared response to the process of birth itself. The majority of unique changes in the lung, however, suggest a lung-specific response likely related to the change in alveolar oxygen content at birth.
The gene exhibiting the greatest change in expression in the lung with birth in room air was the transcription factor Kruppel-like factor 4. Klf4, also known as gut Kruppel-like factor, was initially identified as a tissue-specific gene that inhibited cell proliferation and stimulated differentiation in the gut. The message was highly abundant in colon tissue, particularly in the epithelial cells of the crypts where there is a tight link between growth arrest and differentiation. Klf4 regulates cell proliferation by inhibiting cell cycle progression and inducing growth arrest 
. This function is relevant in murine lung development as mesenchymal and epithelial cell proliferation is known to progressively decline starting late in gestation and continuing into the early postnatal period 
. Transcription factors regulating this process remain poorly defined but our data suggests a role for Klf4. Hence we focused the reminder of our studies on this transcription factor. Klf4 induces growth arrest in cells via the cell cycle inhibitor p21cip1/Waf1
using transcriptional mechanisms to activate the p21cip1/Waf1
promoter and to recruit p53 to this promoter 
. Whether this mechanism is active in the lung fibroblasts or epithelial cells will require further study, but linkage between p21cip1/Waf1
and Klf4 is suggested by the fact that Klf4 deficiency causes a decrease in the level of p21cip1/Waf1
mRNA expression in the late fetal lung. In addition, cell proliferation persists at birth in fibroblasts and airway epithelial cells.
The role of Klf4 in regulating cell differentiation is well described in the epithelium of the skin of the Klf4 null mouse 
, as well as goblet cells of the cornea 
and the colon 
. There is no similar description of Klf4 regulating mesenchymal cell differentiation, even though Klf4 expression has previously been described in the mesenchyme during development 
. Our data clearly shows that connective tissue gene expression for smooth muscle actin, fibronectin, tenascin C and the alpha 1 chain of Type 1 collagen are Klf4 targets in fibroblasts of the developing lung. And in the absence of Klf4 expression, fibroblast connective tissue gene expression is down-regulated in perinatal lung. The mechanism appears transcriptional in nature, but the exact details await further study. Nonetheless, impaired connective tissue gene expression has at least two distinct biological implications for postnatal lung development. First, the newborn lung is easily ruptured under pressure as the connective tissue matrix that forms the lung scaffold is rudimentary at birth. The tensile strength that accumulates in the postnatal period is largely due to collagen synthesis and deposition 
. Lack of tensile strength predisposes to excessive lung compliance and emphysema 
. Second, fibronectin, type 1 collagen and tenascin C expression together with smooth muscle actin defines a specific subpopulation of lung fibroblasts known as the myofibroblast. The cells are known to localize at the tips of secondary saccules and express connective tissue proteins that may be involved with the early postnatal formation of alveoli 
. Loss of smooth muscle actin expression in the absence of Klf4 suggests impaired myofibroblast differentiation which may disrupt alveogenesis 
. Interestingly, smooth muscle actin protein expression in the Klf4 null lung was preserved in vascular smooth muscle cells, which correlates with the known role of Klf4 as a negative regulator of smooth muscle actin gene expression in these cells 
. Our studies show that Klf4 is a positive regulator of smooth muscle actin gene expression in lung myofibroblasts. Therefore Klf4 differentially regulates smooth muscle actin gene expression in these two lung cell types.
The presence of increased cell death in the Klf4 null lung at birth supports a recently defined role for Klf4 as an inhibitor of apoptosis 
. Apoptosis is present even in the normal lung at birth where it is believed to play a physiologic role in postnatal lung development 
. And our microarray data identified apoptosis-related genes as an overrepresented functional gene category among the 157 genes changes that occurred at 2 hours after birth. The mechanism by which Klf4 inhibits apoptosis is not yet fully resolved, though Klf4 can block Bax, a pro-apoptotic protein, in a p53 dependent fashion, and can directly suppress p53 transcription 
. The presence of robust induction of activated p53, p21cip1/Waf1
and caspase-3 in the Klf4 null lung at birth supports this inhibitory role for Klf4 in apoptosis. While the excessive degree of apoptosis in the Klf4 null lung, compared to normal lung, could contribute to loss of gene expression at birth, the absence of apoptosis in vascular smooth muscle cells within large blood vessels indicates that apoptosis was not a general phenomenon, rather it is cell specific.
The magnitude of apoptosis and induction of p21cip1/Waf1
mRNA and protein suggests that the Klf4 null lung may be under stress at birth 
. Stress from oxidants, exposure to heat, chemical and mechanical forces, and nutrient deprivation is known to activate Klf4 expression 
. Our data shows that Klf4 mRNA induction in lung fibroblasts exposed to hyperoxia occurs at a transcriptional level in vitro and is not dependent on protein synthesis. The mechanisms mediating this transcriptional regulation will require further study. That oxidant stress plays a role in the response of Klf4 to oxygen in vivo is suggested by the fact that Klf4 mRNA induction in normal lung at birth is associated with transient oxidant stress, and that both Klf4 mRNA induction and the degree of oxidant stress are attenuated with birth in 10% oxygen. Alveolar oxygen is already known to affect antioxidant, cytokine and transcription factor gene expression at birth 
. Our study is the first to suggest a physiologic role for oxygen as a regulator of a transcription factor that is required for fibroblast and myofibroblast differentiation in normal postnatal lung development ()
Lastly, our Klf4 data in perinatal lung complements existing lung microarray data that found Klf4 to be induced during development 
, post-pneumonectomy 
, and with exposure to cigarette smoke 
and ozone 
. Taken together, Klf4 regulated developmental pathways are likely recapitulated during repair of lung injury. Our study extends the role of Kruppel-like family members in lung development beyond Klf2, lung Kruppel like factor 
, and Klf5 
and shows that Klf4 serves a distinct role in regulating lung myofibroblast connective tissue gene expression. Since the gene targeted Klf4 null mouse dies within 15 hours of birth, ultimate proof of this birth shock hypothesis awaits conditional deletion of Klf4 expression from lung fibroblasts in the perinatal period.