Src Knockout Mice Research Articles

Tyrosine Kinase Src Is a Regulatory Factor of Bone Homeostasis.

Osteoclasts, which resorb the bone, and osteoblasts, which form the bone, are the key cells regulating bone homeostasis. Osteoporosis and other metabolic bone diseases occur when osteoclast-mediated bone resorption is increased and bone formation by osteoblasts is decreased. Analyses of tyrosine kinase Src-knockout mice revealed that Src is essential for bone resorption by osteoclasts and suppresses bone formation by osteoblasts. Src-knockout mice exhibit osteopetrosis. Therefore, Src is a potential target for osteoporosis therapy. However, Src is ubiquitously expressed in many tissues and is involved in various biological processes, such as cell proliferation, growth, and migration. Thus, it is challenging to develop effective osteoporosis therapies targeting Src. To solve this problem, it is necessary to understand the molecular mechanism of Src function in the bone. Src expression and catalytic activity are maintained at high levels in osteoclasts. The high activity of Src is essential for the attachment of osteoclasts to the bone matrix and to resorb the bone by regulating actin-related molecules. Src also inhibits the activity of Runx2, a master regulator of osteoblast differentiation, suppressing bone formation in osteoblasts. In this paper, we introduce the molecular mechanisms of Src in osteoclasts and osteoblasts to explore its potential for bone metabolic disease therapy.

The (Patho)Biology of SRC Kinase in Platelets and Megakaryocytes.

Proto-oncogene tyrosine-protein kinase SRC (SRC), as other members of the SRC family kinases (SFK), plays an important role in regulating signal transduction by different cell surface receptors after changes in the cellular environment. Here, we reviewed the role of SRC in platelets and megakaryocytes (MK). In platelets, inactive closed SRC is coupled to the β subunit of integrin αIIbβ3 while upon fibrinogen binding during platelet activation, αIIbβ3-mediated outside-in signaling is initiated by activation of SRC. Active open SRC now further stimulates many downstream effectors via tyrosine phosphorylation of enzymes, adaptors, and especially cytoskeletal components. Functional platelet studies using SRC knockout mice or broad spectrum SFK inhibitors pointed out that SRC mediates their spreading on fibrinogen. On the other hand, an activating pathological SRC missense variant E527K in humans that causes bleeding inhibits collagen-induced platelet activation while stimulating platelet spreading. The role of SRC in megakaryopoiesis is much less studied. SRC knockout mice have a normal platelet count though studies with SFK inhibitors point out that SRC could interfere with MK polyploidization and proplatelet formation but these inhibitors are not specific. Patients with the SRC E527K variant have thrombocytopenia due to hyperactive SRC that inhibits proplatelet formation after increased spreading of MK on fibrinogen and enhanced formation of podosomes. Studies in humans have contributed significantly to our understanding of SRC signaling in platelets and MK.

Nonphosphorylatable Src Ser75 Mutation Increases Ethanol Preference and Consumption in Mice.

Src is highly expressed in CNS neurons and contributes not only to developmental proliferation and differentiation but also to high-order brain functions, such as those contributing to alcohol consumption. Src knock-out mice exhibit no CNS abnormalities, presumably due to compensation by other Src family kinases (SFKs), but have a shortened lifespan and osteopetrosis-associated defects, impeding investigations of the role of Src on behavior in adult mice. However, the Unique domain of Src differs from those in other SFKs and is phosphorylated by cyclin-dependent kinase 1 (Cdk1) and Cdk5 at Ser75, which influences its postmitotic function in neurons. Therefore, ethanol consumption in mice harboring nonphosphorylatable (Ser75Ala) or phosphomimetic (Ser75Asp) Src mutants was investigated. Mice harboring the Ser75Ala Src mutant, but not the Ser75Asp mutant, had a higher preference for and consumption of solutions containing 5% and 10% ethanol than wild-type mice. However, plasma ethanol concentrations and sensitivities to the sedative effects of ethanol were not different among the groups. In mice harboring the Ser75Ala Src mutant, the activity of Rho-associated kinase (ROCK) in the striatum was significantly lower and Akt Ser473 phosphorylation was significantly higher than in wild-type mice. These results suggest that Src regulates voluntary ethanol drinking in a manner that depends on Ser75 phosphorylation.

A Ser75-to-Asp phospho-mimicking mutation in Src accelerates ageing-related loss of retinal ganglion cells in mice

Src knockout mice show no detectable abnormalities in central nervous system (CNS) post-mitotic neurons, likely reflecting functional compensation by other Src family kinases. Cdk1- or Cdk5-dependent Ser75 phosphorylation in the amino-terminal Unique domain of Src, which shares no homology with other Src family kinases, regulates the stability of active Src. To clarify the roles of Src Ser75 phosphorylation in CNS neurons, we established two types of mutant mice with mutations in Src: phospho-mimicking Ser75Asp (SD) and non-phosphorylatable Ser75Ala (SA). In ageing SD/SD mice, retinal ganglion cell (RGC) number in whole retinas was significantly lower than that in young SD/SD mice in the absence of inflammation and elevated intraocular pressure, resembling the pathogenesis of progressive optic neuropathy. By contrast, SA/SA mice and wild-type (WT) mice exhibited no age-related RGC loss. The age-related retinal RGC number reduction was greater in the peripheral rather than the mid-peripheral region of the retina in SD/SD mice. Furthermore, Rho-associated kinase activity in whole retinas of ageing SD/SD mice was significantly higher than that in young SD/SD mice. These results suggest that Src regulates RGC survival during ageing in a manner that depends on Ser75 phosphorylation.

Lactation failure in Src knockout mice is due to impaired secretory activation

BackgroundMammary gland development culminates in lactation and is orchestrated by numerous stimuli and signaling pathways. The Src family of nonreceptor tyrosine kinases plays a pivotal role in cell signaling. In order to determine if Src plays a role in mammary gland development we have examined mammary gland development and function during pregnancy and lactation in mice in which expression of Src has been eliminated.ResultsWe have characterized a lactation defect in the Src-/- mice which results in the death of over 80% of the litters nursed by Src-/- dams. Mammary gland development during pregnancy appears normal in these mice; however secretory activation does not seem to occur. Serum prolactin levels are normal in Src-/- mice compared to wildtype controls. Expression of the prolactin receptor at both the RNA and protein level was decreased in Src-/- mice following the transition from pregnancy to lactation, as was phosphorylation of STAT5 and expression of milk protein genes. These results suggest that secretory activation, which occurs following parturition, does not occur completely in Src-/- mice. Failed secretory activation results in precocious involution in the mammary glands of Src-/- even when pups were suckling. Involution was accelerated following pup withdrawal perhaps as a result of incomplete secretory activation. In vitro differentiation of mammary epithelial cells from Src-/- mice resulted in diminished production of milk proteins compared to the amount of milk proteins produced by Src+/+ cells, indicating a direct role for Src in regulating the transcription/translation of milk protein genes in mammary epithelial cells.ConclusionSrc is an essential signaling modulator in mammary gland development as Src-/- mice exhibit a block in secretory activation that results in lactation failure and precocious involution. Src appears to be required for increased expression of the prolactin receptor and successful downstream signaling, and alveolar cell organization.

Impairment of Decidualization in SRC-Deficient Mice1

Many signaling events induced by ovarian steroid hormones, cytokines, and growth factors are involved in the process of decidualization of human and rodent endometrium. We have reported previously that tyrosine kinase activation of SRC functionally participates in decidualization of human endometrial stromal cells. To address its essential role in decidualization, we examined, using wild-type and Src knockout mice, whether the process of decidualization was impaired in the absence of SRC. Immunohistochemistry using an antibody specific for the active form of SRC revealed that the active SRC was expressed prominently in the decidualizing stromal cells of the pregnant wild-type mouse. Moreover, the active SRC was upregulated in the uterine horn with artificially stimulated decidual reaction. In comparison with wild-type and Src heterozygous mice, the uterus of Src null mice showed no apparent decidual response following artificial stimulation. Ovarian steroid-induced decidualization in vitro, as determined by morphological changes and expression of decidual/trophoblast prolactin-related protein and prostaglandin-endoperoxide synthase 2 (also known as Cox2), both of which are decidualization markers, did not occur in a timely fashion in endometrial stromal cells isolated from the uteri of SRC-deficient mice compared to those from wild-type and Src heterozygous mice. Our results collectively suggest that SRC is an indispensable signaling component for maximal decidualization in mice.

Alterations in Reproductive Function in Src Tyrosine Kinase Knockout Mice

The role of Src tyrosine kinase in regulating reproductive processes in female mice was investigated using Src wild-type (+/+), heterozygous (+/-), and knockout (-/-) mice. Ovarian Src kinase activity transiently increased in Src +/+ mice following eCG administration. Src knockout mice were infertile. Estrous cycles and vaginal opening in Src knockouts were variable and altered compared with Src +/+ and +/- mice. Follicle development was compromised in Src knockout mice as evidenced by reduced numbers of large pre-antral and antral follicles compared to Src +/+ mice. Corpora lutea were not observed in the ovaries of Src knockout mice; however, administration of eCG and hCG did result in ovulation. Serum LH and FSH on d 40 and 52 of age did not differ between Src wild-type and knockout females. Results from these studies reveal that female Src knockout mice are infertile due to reduced follicle development and anovulation.

Functional significance of human trp1 and trp3 in store-operated Ca(2+) entry in HEK-293 cells.

The Drosophila trp (transient receptor potential) gene appears to encode the Drosophila store-operated channel (SOC), and some mammalian trp homologues have been proposed to encode mammalian SOCs. This study provides evidence for the expression of three trp homologues (Mtrp2, Mtrp3, and Mtrp4) in fibroblasts from wild-type and src knockout mice, and four trp homologues (Htrp1, Htrp3, Htrp4, and Htrp6) in human embryonic kidney (HEK-293) cells based on RT-PCR techniques. In HEK-293 cells stably transfected with a 323-bp Htrp3 antisense construct (Htrp3AS), Northern blot analysis revealed that the expression of a 4-kb transcript was dramatically suppressed in comparison to that observed in cells stably transfected with a short Htrp3 sense construct (Htrp3S). Activity of SOCs, monitored as Ba(2+) influx following Ca(2+) store depletion with thapsigargin, was reduced by 32% in Htrp3AS cells in comparison with Htrp3S cells. Transient transfection of a 369-bp Htrp1 antisense construct in cells stably expressing Htrp3AS induced a higher level of inhibition (55%) of store-operated Ca(2+) entry. These data suggest that Htrp1 and Htrp3 may be functional subunits of SOCs.

Src is required for cell migration and shape changes induced by fibroblast growth factor 1.

Fibroblast growth factor 1 (FGF-1) is a potent chemotactic factor and induces tyrosine phosphorylation of a cortical actin-associated protein (cortactin). The tyrosine phosphorylation of cortactin induced by FGF-1 requires the tyrosine residues 421, 482 and 466, which are targeted by the protein tyrosine kinase Src in vitro. Furthermore, FGF-1 is unable to induce tyrosine phosphorylation of cortactin within the cells derived from Src knockout mice (Src-/-), indicating that Src is required for the tyrosine phosphorylation of cortactin induced by FGF-1. Although Src-/- cells are able to undergo rapid proliferation, they are impaired to respond to FGF-1 for the shape change and cell migration. Morphological analysis further reveals that FGF-1 fails to induce the formation of polarized lamellipodia and the translocation of cortactin into the leading edge of Src-/- cells. Consistent with the mitogenic response to FGF-1, the lack of Src does not affect the tyrosine phosphorylation of Snt (or Frs2), a FGF-1 early signaling protein that links to Ras. Therefore, our data support the notion that Src and cortactin participate in a FGF signal pathway for cell migration and shape change rather than mitogenesis.