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Absorption, distribution, metabolism, and excretion properties of a small interfering RNA (siRNA) formulated in a lipid nanoparticle (LNP) vehicle were determined in male CD-1 mice following a single intravenous administration of LNP-formulated [(3)H]-SSB siRNA, at a target dose of 2.5 mg/kg. Tissue distribution of the [(3)H]-SSB siRNA was determined using quantitative whole-body autoradiography, and the biostability was determined by both liquid chromatography mass spectrometry (LC-MS) with radiodetection and reverse-transcriptase polymerase chain reaction techniques. Furthermore, the pharmacokinetics and distribution of the cationic lipid (one of the main excipients of the LNP vehicle) were investigated by LC-MS and matrix-assisted laser desorption ionization mass spectrometry imaging techniques, respectively. Following i.v. administration of [(3)H]-SSB siRNA in the LNP vehicle, the concentration of parent guide strand could be determined up to 168 hours p.d. (post dose), which was ascribed to the use of the vehicle. This was significantly longer than what was observed after i.v. administration of the unformulated [(3)H]-SSB siRNA, where no intact parent guide strand could be observed 5 minutes post dosing. The disposition of the siRNA was determined by the pharmacokinetics of the formulated LNP vehicle itself. In this study, the radioactivity was widely distributed throughout the body, and the total radioactivity concentration was determined in selected tissues. The highest concentrations of radioactivity were found in the spleen, liver, esophagus, stomach, adrenal, and seminal vesicle wall. In conclusion, the LNP vehicle was found to drive the kinetics and biodistribution of the SSB siRNA. The renal clearance was significantly reduced and its exposure in plasma significantly increased compared with the unformulated [(3)H]-SSB siRNA.

The epidermal growth factor receptor family member ERBB4 is required for mammary gland development and lactation. ERBB4 activities in the breast are mediated through the signal transducer and activator of transcription (STAT) family member STAT5A, and ERBB4 directly activates STAT5A, in part, through phosphorylation of STAT5A at the regulatory Tyr-694. Here we show that STAT5A regulation by ERBB4 is also mediated through STAT5A serine phosphorylation. Using a reverse-phase high performance liquid chromatography tandem mass spectrometry analysis of proteolytically digested STAT5A coexpressed with ERBB4, we identified STAT5A serine phosphorylations at the previously described Ser-779 and at the novel Ser-127/Ser-128. Immunohistochemistry of wild-type and ERBB4-null mammary glands at late pregnancy showed that ERBB4 expression was required for STAT5A phosphorylation at Ser-779. Independent serine-to-alanine residue substitutions in full-length STAT5A revealed that although STAT5A Ser-779 phosphorylation was dispensable for phosphorylation of STAT5A at Tyr-694 and subsequent DNA binding, Ser-779 was required to stabilize an interaction with ERBB4 and mediate ERBB4-induced STAT5A stimulation of gene expression. STAT5A Ser-127/Ser-128, on the other hand, was required for ERBB4-induced phosphorylation of Tyr-694, whereas Ser-779 and as yet unidentified tyrosine residues were phosphorylated in the absence of Ser-127/Ser-128. In addition, STAT5A S127A/S128A remained associated with ERBB4 but failed to bind DNA or activate transcription in response to ERBB4 coexpression. Our studies demonstrate that phosphorylation of STAT5A at Ser-127/Ser-128 and Ser-779 are obligatory events regulating ERBB4-mediated activation of STAT5A.

The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr694 in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863–31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser779, located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser779-specific antiserum, we have determined that Ser779 is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser779. Ser725 in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218–30224). Here we show that mutagenesis of Ser725, Ser779, or a combination of Ser725/779 to an Ala had no effect on prolactin-induced transcriptional activation of a β-casein reporter construct. However, following prolactin induction the Ser725 mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser725 phosphorylation has an impact on signal duration.

Epidermal growth factor (EGF) binding to its receptor, ErbB1, triggers various signal transduction pathways, one of which leads to the activation of signal transducer and activator of transcription (Stat) factors. The mechanism underlying ErbB1-induced Stat activation and whether Stats are downstream targets of other ErbB receptors have not been explored. In this report we show that ErbB2, ErbB3, and ErbB4 do not potentiate Stat5 phosphorylation by EGF. However, neu differentiation factor-induced heterodimers of ErbB2 and ErbB4 activated Stat5. In A431 cells, Stat1, Stat3, and Stat5, were constitutively complexed with ErbB1 and rapidly phosphorylated on tyrosine in response to EGF. Neither mutation of the conserved tyrosine residue (Tyr694) nor inactivation of the Stat5a SH2 domain disrupted this association. However, an intact SH2 domain was necessary for EGF-induced Stat5a phosphorylation. In contrast to prolactin, which induced only Tyr694 phosphorylation of Stat5a, EGF promoted phosphorylation on Tyr694 and additional tyrosine residue(s). Janus kinases (Jaks) were also constitutively associated with ErbB receptors and were phosphorylated in response to EGF-related ligands. However, we provide evidence that EGF- and neu differentiation factor-induced Stat activation are dependent on Src but not Jak kinases. Upon EGF stimulation, c-Src was rapidly recruited to Stat/ErbB receptor complexes. Pharmacological Src kinase inhibitors and a dominant negative c-Src ablated both Stat and Jak tyrosine phosphorylation. However, dominant negative Jaks did not affect EGF-induced Stat phosphorylation. Taken together, the experiments establish two independent roles for Src kinases: (i) key molecules in ErbB receptor-mediated Stat signaling and (ii) potential upstream regulators of Jak kinases.