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During embryonic life, the lungs are actually unnecessary as breathing organs; however they must be developed enough to begin functioning immediately after birth. Hence, optimal postnatal lung function is reliant on a tightly controlled embryonic lung development program. This developmental program has been divided into five stages: embryonic, psuedogladular, canalicular, terminal saccular, and alveolar.
The embryonic stage begins at embryonic day (E)9.5 in the mouse (~ 3 weeks in human pregnancy), with the formation of trachea from the foregut endoderm, which is followed quickly by two primordial lung buds. During the pseudoglandular stage (E9.5 - 16.5 in the mouse, 5 - 17 weeks in humans) the lung undergoes much of its branching morphogenesis, and this is followed by the canalicular phase (E16.5 - 17.5 in mouse, 16 - 25 weeks in humans) where growth of the lung is accompanied by the organisation of the vasculature along the airway. The terminal saccular stage (E17.5 - postnatal (P)5 in mouse, 24 weeks to late fetal period in humans) starts the thinning of airway walls, and the enlargement of peripheral airways and gas-exchange surface areas to enable to the lung to start supporting air exchange, if required. Finally, the alveolar stage (P5 - P30 in mouse, late fetal period to childhood in humans) sees the formation of the alveoli which forms the majority of the gas exchange surface (1, 2).
Branching morphogenesis, which occurs mainly in the psuedoglandular stage, is a crucial part of lung development. Despite the apparent complexities of lung branching, the budding patterns at early stages take place through only three simple genetically encoded modes of branching, termed domain branching, planar bifurcation, and orthogonal bifurcation (1, 3, 4), which are then continuously repeated to form different compositions of branches. As these processes are genetically encoded, they are reliant on interactions with various intrinsic factors, transcription factors and proteins (reviewed in depth by Warburton et al. (5)), and disruptions to these interactions, as well as the influence of various extrinsic factors, can have negative effects on the lung development programme - leading to lungs that have impaired function (6).
A number of studies have pointed towards Ca2+ playing a role in lung development. In 1995, Roman demonstrated that the L-type calcium channel blocker nifedipine affected lung branching morphogenesis and abolished the appearance of spontaneous airway contractions (7), suggestive that calcium ion transport was necessary for the normal development of the lung. Further work by Jesudason et al. confirmed these initial observations and demonstrated that the presence of nifedipine abolished both the peristaltic smooth muscle contractions and lung growth mediated by Fibroblast Growth Factor-10 (FGF-10) (8), a critical intrinsic factor for the development of the mammalian lung (9).
These peristaltic movements, regulated by pacemaker areas within the proximal airway (8), lead to waves of fluid being directed to the distal lung bud and allow regulation of the pressure through rhythmic stretch and relaxation (10). The frequency of these waves is also associated with the rates of lung growth, with increased frequencies of peristaltic waves associated with enhanced lung growth, and vice versa (8). [Ca2+]i-imaging experiments revealed that airway contractions immediately follow spontaneous and regenerative [Ca2+]i waves in the airway smooth muscle cells, and that these are dependent on both extra- and intra-cellular Ca2+ (11). Interestingly, it has also been shown that these [Ca2+]i waves are abnormal in experimental models of lung hypoplasia (12).
However, little was known about the effects of [Ca2+]o as an extrinsic factor on lung development. This is an interesting situation, as many laboratories routinely perform their lung explant cultures in media with widely differing [Ca2+]o conditions (e.g. 1.05 mM [Ca]2+o or 2.50 mM [Ca]2+o) to that seen in the fetus (~ 1.70 mM [Ca2+]o) (13). Partly this is due to the fact that previous studies have not provided a mechanism for [Ca2+]o sensing within the lung, with expression of the CaSR not observed in either adult bovine or rat lungs (14, 15). However, more recently, the CaSR has been shown to be expressed in the fetal lungs of both humans and mice, mainly during the pseudoglandular stage [Figure?] (16, 17).
In mouse fetal lungs, the expression of the CaSR is developmentally regulated and can be detected using immunohistochemical methods from as early as E10.5 in the mouse. Expression of the CaSR is initially demonstrated in the epithelium; however, from E13.5 it also starts to appear within the mesenchyme. Expression of the CaSR within the mouse lung is maintained until at least E18.5, however, is absent in newborn and adult mouse lung (16).
It has also been demonstrated, using mouse E12.5 lung explant cultures (18, 19), that lung branching is sensitive to extracellular calcium over the range 0.5 - 2.0 mM (16). Interestingly, higher concentrations of [Ca2+]o, similar to that seen in the fetal extracellular milieu (~ 1.70 mM), have a suppressive effect on branching over 48 h (16). Furthermore, this effect is mimicked in lung cultured in the presence of the CaSR-specific calcimimetic, NPS R-568, at 1.05 mM [Ca2+]o, indicative of the fact that the CaSR may play an important role in regulating branching morphogenesis (16).
Similar to many other CaSR-expressing cells types (20, 21), exposure of isolated E12.5 epithelium lung buds to increases in extracellular Ca2+o and NPS R-568 leads to increases in phospholipase C (PLC)-evoked [Ca2+]i release from internal calcium stores (16). In addition, inhibition of PLC with U73122 blocked the effect of high (1.70 mM) [Ca2+]o on lung branching morphogenesis, restoring branching levels to that seen in the presence of low (1.05 mM) [Ca2+]o. Phosphoinositide 3-kinase (PI3K) also appears to be an important downstream component for CaSR-mediated regulation of lung branching, with the PI3K inhibitor, LY294002, similarly reversing the effect of high Ca2+o and calcimimetics on branching (16). Interesting, blockers of the mitogen-activated protein kinase (MAPK) family, another common CaSR-activated signalling protein, failed to have an effect (16).
The reduction in lung branching caused by high [Ca2+]o and calcimimetics appears to be caused by CaSR-induced reduction of cellular proliferation. The CaSR has been implicated in the control of proliferation in a number of different cells types including parathyroid cells (22, 23) and colonic epithelial cells (24). Culturing E12.5 lung explants in the presence of 1.70 mM Ca2+o approximately halved the number of phospho-histone H3-positive cells (16) - used as a marker of mitosis (25). However, there were no significant differences in the number of apoptotic cells. Therefore, it seems that CaSR-mediated regulation of the lung branching programme occurs through the control of cellular proliferation and requires the release of Ca2+i via PLC and activation of PI3K.
Adequate embryonic lung growth also requires fluid-dependent lung distension (26). Interestingly, it has been observed that E12.5 embryonic lungs cultured in high [Ca2+]o or in the presence of calcimimetics show an increase in luminal fluid volume and greater distension (16), suggesting an increase in fluid secretion.
Within the embryo, the lungs are filled with fluid secreted from cells in the lung epithelium into the developing lumen. Fluid secretion within the embryonic lung is driven by secondary active Cl- transport (27), with the basolateral entry of Cl- into the epithelium occurring via the Na-K-Cl cotransporter (NKCC) (28, 29). This then facilitates apical fluid secretion from apical Cl- channels (30), however, despite the expression of a number of Cl- channels within the lung, including the cystic fibrosis conductance regulator (CFTR) (31) and members of the Chloride Channel (CLC) family (32), the exact identity of the channel involved remains unclear.
This secretion of Cl- into lung lumen leads to a negative transepithelial potential difference (PD), which can be then measured using electrophysiological techniques and reflects the rate of fluid secretion (16). Embryonic lungs cultured in fetal/high [Ca2+]o conditions showed a significantly negative PD, in comparison than those cultured in adult/low [Ca2+]o. Furthermore, activation of the CaSR with NPS R-568 also led to significantly higher negative PD, indicative of CaSR-mediated increases in fluid secretion (16). These results suggest that activation of the CaSR may stimulate transepithelial transport, possibly through interactions with either NKCC or one of the apical chloride channels, however the mechanism is still unknown.
It has been previously suggested that accumulation of fluid, leading to distension of the lung, may act as a negative block on lung branching (33). However, in contrast to this, 'rescue' experiments where lungs were cultured for 24h in either 1.05 mM or 1.70 mM [Ca2+]o before being switched, suggests that fluid secretion is not the primary inhibiting mechanism of lung branching (16). Switching lung explants from high [Ca2+]o to low [Ca2+]o after 24 h completely abrogated the suppressive effect of high [Ca2+]o on lung branching. However, it had no effect on transepithelial PD, with PD values showing no significant change from lungs cultured in high [Ca2+]o for 48 h (16).
While it may not be the primary inhibiting factor, luminal fluid does play an important role in the lung branching pattern, through a process called viscous fingering. The resulting increase in branching occurs through increases in mechanical pressure, wherein the less viscous fluid within the branching epithelial tubes of the lung infiltrates the more viscous fluid within the surrounding mesenchymal matrix (34). Therefore, if the pressure remains constant there would be no relative change in branching. This process relies heavily on the cellular structures that surround that fluid space, as structural integrity is required to maintain osmotic pressure with high pressure in the lumen (35). It is possible that activation of the CaSR may affect this process with increased Cl- secretion leading to a decrease in viscosity that does not affect pressure within the lumen, or alternatively, increased Cl- secretion is coupled with changes in cellular structure which do not allow viscous fingering of lung branching to occur.
Greater understanding of the mechanisms that regulate lung branching are important, as there is a need for the lung to control the number of terminal branches for optimal alveoli development. A number of different factors, including abnormal signalling and/or regulation of growth factors, and exposure to oxygen; can all adversely affect the lung developmental programme (1, 5, 36). Furthermore, as the pulmonary vasculature develops in symmetry with branching morphogenesis, these factors can have profound effects on the lung structure, e.g. hypoplasia, leading to a deficiency of gas exchange and possible neonatal respiratory failure. Even more subtle lung dysplasia, which does not lead to neonatal death, may still lead to predisposition for respiratory diseases later in life, such as early onset chronic obstructive pulmonary disease (COPD) (1, 36).
The work by Finney et al. detailed above suggests that [Ca2+]o, acting via the CaSR, may act as an extrinsic factor that regulates lung branching to ensure an optimal number of branches is achieved (16). In addition to suppressing lung branching morphogenesis, [Ca2+]o works to stimulate fluid secretion, possibly to enable lung development to continue simultaneously with optimal lumen distension.
Furthermore, the 'rescue' experiments also open up an interesting possibility that pharmacological modulators of the CaSR may be able to manipulate defective lung branching. While switching the lungs for high [Ca2+]o to low [Ca2+]o was able to reverse the inhibition of the branching programme, the opposite experiment (switching from low [Ca2+]o after 24h to high [Ca2+]o) had no effect - with the lungs showing a high number of branches similar to that seen in lungs cultured in 1.05 mM Ca2+o for 48 h (16). These results suggest that once the lung branching programme is switched on it may be difficult to be stopped; however, it may be possible to force underdeveloped lungs into branching if they are exposed to more favourable conditions.
These results may also help explain observed respiratory problems in patients and mice with mutations in the CaSR. Inactivating mutations of the CaSR have previously been linked with chronic and interstitial lung disease and a reduction in the ability to transport gas into and out of the blood (37-39). It is possible that in these cases the hypercalcemic conditions in the fetus were unable to control excessive proliferation; leading to hyperplastic lungs with may underlie these conditions (16). Conversely, activating mutations of the CaSR have been linked with ectopic calcifications in the mouse lung (40) and recurring upper respiratory tract infections in humans (41). However, further studies are required to examine the incidence of respiratory pathologies in populations of patients with inactivating/activating mutations of the CaSR.
An attempt has been made to examine the effect of ablation of the CaSR in the development of the lung, using the exon 5-deleted CaSR knockout mouse model (CaSREx5-/-) (42, 43). However, as has been previously reported in keratinocytes (44, 45) and growth plate chrondrocytes (46), knocking out the full length CaSR induced the expression of a fully functional exon 5-less spliced variant which effectively rescued the phenotype (43). Lung explants from E12.5 CaSREx5-/- mice are able to respond to high [Ca2+]o and CaSR selective concentrations of NPS R-568, suppressing lung branching morphogenesis in a similar fashion to that previously reported (16), and their wildtype CaSREx5+/+ littermates.
These results mean that further studies are still required to examine the effect of aberrant calcium-sensing receptor function on lung development and its potential post-natal consequences to help conclusively define the role of the CaSR in lung development. However, it appears that both [Ca2+]o and the CaSR are important regulating factors in the development of the lung, helping to suppress lung branching morphogenesis while stimulating fluid secretion to enable optimal lumen distension (16). In addition, these results suggest there is a possibility that pharmacological modulators of the CaSR, such as the already FDA approved cinacalcet, may be potential novel treatments for hypo- or hyper-plastic lungs.