SHP-1 Knockdown Research Articles

Antagonizing roles of SHP1 in the pathogenesis of Helicobacterpylori infection.

SHP1 has been documented as a tumor suppressor and it was thought to play an antagonistic role in the pathogenesis of Helicobacter pylori infection. In this study, the exact mechanism of this antagonistic action was studied. AGS, MGC803, and GES-1 cells were infected with H. pylori, intracellular distribution changes of SHP1 were first detected by immunofluorescence. SHP1 overexpression and knockdown were then constructed in these cells to investigate its antagonistic roles in H. pylori infection. Migration and invasion of infected cells were detected by transwell assay, secretion of IL-8 was examined via ELISA, the cells with hummingbird-like alteration were determined by microexamination, and activation of JAK2/STAT3, PI3K/Akt, and ERK pathways were detected by immunoblotting. Mice infection model was established and gastric pathological changes were evaluated. Finally, the SHP1 activator sorafenib was used to analyze the attenuating effect of SHP1 activation on H. pylori pathogenesis invitro and invivo. The sub-localization of SHP1 changed after H. pylori infection, specifically that the majority of the cytoplasmic SHP1 was transferred to the cell membrane. SHP1 inhibited H. pylori-induced activation of JAK2/STAT3 pathway, PI3K/Akt pathway, nuclear translocation of NF-κB, and then reduced EMT, migration, invasion, and IL-8 secretion. In addition, SHP1 inhibited the formation of CagA-SHP2 complex by dephosphorylating phosphorylated CagA, reduced ERK phosphorylation and the formation of CagA-dependent hummingbird-like cells. In the mice infection model, gastric pathological changes were observed and increased IL-8 secretion, indicators of cell proliferation and EMT progression were also detected. By activating SHP1 with sorafenib, a significant curative effect against H. pylori infection was obtained invitro and invivo. SHP1 plays an antagonistic role in H. pylori pathogenesis by inhibiting JAK2/STAT3 and PI3K/Akt pathways, NF-κB nuclear translocation, and CagA phosphorylation, thereby reducing cell EMT, migration, invasion, IL-8 secretion, and hummingbird-like changes.

SHP-1 knockdown suppresses mitochondrial biogenesis and aggravates mitochondria-dependent apoptosis induced by all trans retinal through the STING/AMPK pathways

BackgroundOxidative stress-caused damage to the retinal pigment epithelium (RPE) underlies the onset and progression of age-related macular degeneration (AMD). Impaired mitochondrial biogenesis sensitizes RPE cells to mitochondrial dysfunction, energy insufficiency and death. Src-homology 2 domain-containing phosphatase (SHP)-1 is important in regulating immune responses and cell survival. However, its roles in cell survival are not always consistent. Until now, the effects of SHP-1 on RPE dysfunction, especially mitochondrial homeostasis, remain to be elucidated. We sought to clarify the effects of SHP-1 in RPE cells in response to atRAL-induced oxidative stress and determine the regulatory mechanisms involved.MethodsIn the all trans retinal (atRAL)-induced oxidative stress model, we used the vector of lentivirus to knockdown the expression of SHP-1 in ARPE-19 cells. CCK-8 assay, Annexin V/PI staining and JC-1 staining were utilized to determine the cell viability, cell apoptosis and mitochondrial membrane potential. We also used immunoprecipitation to examine the ubiquitination modification of stimulator of interferon genes (STING) and its interaction with SHP-1. The expression levels of mitochondrial marker, proteins related to mitochondrial biogenesis, and signaling molecules involved were examined by western blotting analysis.ResultsWe found that SHP-1 knockdown predisposed RPE cells to apoptosis, aggravated mitochondrial damage, and repressed mitochondrial biogenesis after treatment with atRAL. Immunofluoresent staining and immunoprecipitation analysis confirmed that SHP-1 interacted with the endoplasmic reticulum-resident STING and suppressed K63-linked ubiquitination and activation of STING. Inhibition of STING with the specific antagonist H151 attenuated the effects of SHP-1 knockdown on mitochondrial biogenesis and oxidative damage. The adenosine monophosphate-activated protein kinase (AMPK) pathway acted as the crucial downstream target of STING and was involved in the regulatory processes.ConclusionsThese findings suggest that SHP-1 knockdown potentiates STING overactivation and represses mitochondrial biogenesis and cell survival, at least in part by blocking the AMPK pathway in RPE cells. Therefore, restoring mitochondrial health by regulating SHP-1 in RPE cells may be a potential therapeutic strategy for degenerative retinal diseases including AMD.

Morin Disrupts Cytoskeleton Reorganization in Osteoclasts through an ROS/SHP1/c-Src Axis and Grants Protection from LPS-Induced Bone Loss.

Morin is a naturally occurring flavonoid with anti-inflammatory and antioxidative properties. Therefore, we hypothesized that morin may prevent inflammatory bone loss by reducing oxidative stress. To investigate the effect of morin on inflammatory bone loss, mice were injected with lipopolysaccharide (LPS). Osteoclasts (OCs) were analyzed by tartrate-resistant acid phosphatase (TRAP) staining and actin ring formation. Micro-computerized tomography analysis indicated that morin prevented LPS-induced bone loss in mice. In vivo TRAP staining indicated that morin decreased the number and surface of the OCs that were increased in LPS-treated mice. Furthermore, in vitro experiments indicated that morin decreased the number and activity of OCs upon LPS stimulation. Morin decreased actin ring-containing OCs with decreased activation of c-Src (Y416)/vav guanine nucleotide exchange factor 3/Ras-related C3 botulinum toxin substrate 1 compared with LPS alone. Morin decreased cytosolic reactive oxygen species (ROS), thus preventing the oxidation of Src homology region 2 domain-containing phosphatase 1 (SHP-1), followed by the inactivation of c-Src via direct interaction with SHP1. Conversely, SHP1 knockdown abolished the inhibitory effect of morin on OCs. Therefore, our findings suggest that morin disrupted cytoskeletal reorganization via an ROS/SHP1/c-Src axis in OCs, thereby granting protection from LPS-induced bone loss, which demonstrates its therapeutic potential against inflammatory bone loss.

SHP-1 acts as a tumor suppressor by interacting with EGFR and predicts the prognosis of human breast cancer.

Objective:The aims of this study were to examine the prognostic value of SHP-1 in breast cancer, its roles in the regulation of breast cancer cell growth and metastasis, and the underlying mechanisms.Methods:Tumor specimens from 160 patients with breast cancer and 160 noncancerous tissues were used to examine the expression of SHP-1 and to analyze its association with overall survival through Kaplan–Meier and multivariate Cox regression analyses. RNA sequencing data and the expression and clinical importance of SHP-1 in breast cancer were evaluated with data from The Cancer Genome Atlas. In vitro and in vivo assays were performed to elucidate the effects of SHP-1 on breast cancer cell proliferation and invasion. Confocal immunofluorescence and GST pulldown assays were used to demonstrate the interaction between SHP-1 and epidermal growth factor receptor, as well as its downstream pathways. Immunohistochemistry and The Cancer Genome Atlas database were used to investigate the clinical association between SHP-1 and EGFR in human breast cancer.Results:SHP-1 expression was associated with better survival in patients with breast cancer, whereas SHP-1 expression was negatively correlated with EGFR in human breast cancer. Ectopic SHP-1 expression significantly suppressed breast cancer cell proliferation, migration, and invasion. SHP-1 knockdown induced a more invasive phenotype and accelerated cell growth. Mechanistically, EGFR, a protein directly interacting with SHP-1, mediates the SHP-1-induced inactivation of Ras/Erk/GSK3β signaling and its downstream effectors.Conclusions:SHP-1 is an important prognostic biomarker in patients with breast cancer, and the SHP-1-EGFR axis is a promising target for treatment.

Inhibition of SHP-1 Expands the Repertoire of Antitumor T Cells Available to Respond to Immune Checkpoint Blockade.

The presence and activity of CD8+ T cells within the tumor microenvironment are essential for the control of tumor growth. Utilizing B16-F10 melanoma tumors that express altered peptide ligands of chicken ovalbumin, OVA257-264, we measured high- and low-affinity OVA-specific responses following adoptive transfer of OT-I CD8+ T cell into mice subsequently challenged with tumors. T-cell receptor (TCR) affinity positively correlated with the frequency of OT-I tumor-infiltrating lymphocytes (TIL). Differences in TCR affinity inversely corresponded to in vivo tumor growth rate. Blockade of the PD-1 and CTLA-4 checkpoints preferentially increased the frequency and antitumor function of TIL responding to high-affinity antigens, while failing to enhance the antitumor activity of low-affinity T cells. To determine whether lowering the TCR activation threshold could enhance the breadth and magnitude of the antitumor T-cell response, we inhibited Src homology region 2 domain-containing phosphatase 1 (SHP-1) in OT-I T cells prior to tumor antigen exposure. SHP-1 knockdown increased the cytokine-producing potential of high- and low-affinity T cells but failed to enhance control of tumor growth. In contrast, when SHP-1 knockdown of OT-I T cells was combined with immunotherapy, we observed a significant and long-lasting suppression of tumor growth mediated by low-affinity T cells. We conclude that lowering the TCR activation threshold by targeting SHP-1 expands the repertoire of T cells available to respond to conventional checkpoint blockade, leading to enhanced control of tumor growth.

Inhibition of SHP-1 expands the repertoire of anti-tumor T cells available to respond to checkpoint blockade therapy

Abstract The presence and activity of CD8+T cells within the tumor microenvironment is essential for the control of tumor growth and impediment of cancer progression. Utilizing B16 F10 melanoma tumors that express altered peptide ligands of OVA257–264, we measured high- and low-affinity OVA-specific responses following adoptive transfer of OT-1 CD8+T cell into tumor-bearing mice. As expected, we found that TCR affinity positively correlated with the frequency of OT-1 tumor infiltrating lymphocytes (TILs). The differences in TCR affinity inversely corresponded to in vivo tumor growth rate. We next sought to determine whether checkpoint blockade therapy (CB: anti-PD-1; anti-CTLA-4) could enhance the recruitment of low affinity T cells. However, CB only increased the frequency of OT-1 TILs responding to high affinity antigens, while failing to alter the response or protective capacity of intermediate to low affinity T cell responses. To explore whether lowering TCR activation threshold could enhance the breadth and magnitude of the anti-tumor T cell response, we used shRNA to knock down Src homology region 2 domain-containing phosphatase 1 (SHP-1) in OT-1 T cells prior to tumor antigen exposure. Unlike CB, SHP-1 knockdown increased the magnitude and effector function of both low affinity and high affinity OT-1 T cells. When SHP-1 knockdown was combined with immunotherapy, we observed an additional boost in the OT-1 response, as well as enhanced tumor control by intermediate to low-affinity OT-1s. We conclude that targeting TCR activation threshold expands the repertoire of T cells available to respond to conventional CB, leading to enhanced control of tumor growth.

SHP-1 inhibits renal ischemia reperfusion injury via dephosphorylating ASK1 and suppressing apoptosis

Apoptosis of tubular epithelium cells (TECs) plays critical roles in renal ischemia reperfusion (I/R) injury, but the molecular regulatory mechanisms of apoptosis still require further investigation. Recently, phosphatase family members have been suggested to regulate multiple aspects of the injury and regeneration response. However, the roles of SHP-1, an important protein-tyrosine phosphatase, in the regulation of renal I/R injury remain unknown. Here, we found that SHP-1 knockdown in vivo significantly increased renal I/R injury and aggravated the apoptosis of TECs. Consistently, after SHP-1 knockdown in TECs in vitro, a sharp increase of apoptosis induced by cobalt dichloride was found. The protective role of SHP-1 was also validated in a TEC cell line stably overexpressing SHP-1. Mechanistically, the ASK1/MKK4/JNK pro-apoptosis signal was over activated after SHP-1 knockdown, and SHP-1 could bind to and dephosphorylate ASK1 to inhibit its activation, thus repressing apoptosis.

Two novel SHP-1 agonists, SC-43 and SC-78, are more potent than regorafenib in suppressing the in vitro stemness of human colorectal cancer cells

Signal transducer and activator of transcription 3 (STAT3) has been shown to play a critical role in the maintenance of cancer stem cells (CSCs). Hence, the inhibition of STAT3 signaling has been suggested to be a viable therapeutic approach for cancers. Moreover, the efficacy of combinations of chemotherapeutic drugs and napabucasin, a small-molecule STAT3 inhibitor, have been assessed in various clinical trials, including those involving patients with metastatic colorectal cancer (CRC).Two recently developed small-molecule STAT3 inhibitors, SC-43 and SC-78, which can stimulate SHP-1 to inactivate STAT3, were found to have anti-tumor activity. In this study, the inhibitory effects of SC-43, SC-78, and regorafenib (a reference drug) on cell viability, STAT3 phosphorylation, and various stemness properties [e.g., sphere-forming and soft agar colony-forming abilities, CD133+/CD44+ (stem cell-like) subpopulations, and the expression of several CSC markers] were examined for both HCT-116 and HT-29 human CRC cells. We found that SC-43 and SC-78 but not regorafenib inhibited constitutive and IL-6-induced STAT3 phosphorylation in HCT-116 and HT-29 cells, respectively. Moreover, SC-43 and SC-78 were more potent than regorafenib in suppressing the stemness properties (except stem cell-like subpopulations) of these cells. As expected, SHP-1 knockdown almost completely abolished the suppressive effects of SC-43 and SC-78 on the sphere formation in both cell lines. Furthermore, SC-43 and SC-78 showed synergistic inhibitory effects with oxaliplatin and/or irinotecan on sphere formation. Overall, our results suggest that SC-43 and SC-78 are potent STAT3 inhibitors that may potentially be used in combination therapy for CRC.

Shp1 positively regulates EGFR signaling by controlling EGFR protein expression in mammary epithelial cells

SH2-domain containing protein tyrosine phosphatase 1 (Shp1/PTPN6) is mainly expressed in hematopoietic cells and acts a negative signaling regulator. Although Shp1 is also expressed in epithelial cells, the function of shp1 in normal epithelial is still less well understood, especially in regulating the growth of epithelial cells. In this study, different shRNAs and siRNAs against Shp1 were used to knockdown Shp1 expression in MCF10A, an immortalized mammary epithelial cell line. Shp1 knockdown resulted in inhibited cell growth in part due to lower percentage of MCF10A cells entering into S phase and reduced cyclin D1 expression. Accordingly, EGF-induced tyrosyl phosphorylation of EGFR and Stat5 was significantly inhibited in cells with Shp1 knockdown compared with control whereas EGF-induced Akt and Erk phosphorylation was not affected by Shp1 knockdown. Further analysis revealed that Shp1 knockdown lead to decreased EGFR protein expression without affecting EGFR mRNA expression or increasing EGFR protein degradation. Our data indicate that Shp1 functions as a positive regulator and acts in a novel mechanism through promoting EGFR protein expression in mammary epithelial cells.

The tyrosine phosphatase SHP-1 regulates hypoxia inducible factor-1α (HIF-1α) protein levels in endothelial cells under hypoxia.

IntroductionThe tyrosine phosphatase SHP-1 negatively influences endothelial function, such as VEGF signaling and reactive oxygen species (ROS) formation, and has been shown to influence angiogenesis during tissue ischemia. In ischemic tissues, hypoxia induced angiogenesis is crucial for restoring oxygen supply. However, the exact mechanism how SHP-1 affects endothelial function during ischemia or hypoxia remains unclear. We performed in vitro endothelial cell culture experiments to characterize the role of SHP-1 during hypoxia.ResultsSHP-1 knock-down by specific antisense oligodesoxynucleotides (AS-Odn) increased cell growth as well as VEGF synthesis and secretion during 24 hours of hypoxia compared to control AS-Odn. This was prevented by HIF-1α inhibition (echinomycin and apigenin). SHP-1 knock-down as well as overexpression of a catalytically inactive SHP-1 (SHP-1 CS) further enhanced HIF-1α protein levels, whereas overexpression of a constitutively active SHP-1 (SHP-1 E74A) resulted in decreased HIF-1α levels during hypoxia, compared to wildtype SHP-1. Proteasome inhibition (MG132) returned HIF-1α levels to control or wildtype levels respectively in these cells. SHP-1 silencing did not alter HIF-1α mRNA levels. Finally, under hypoxic conditions SHP-1 knock-down enhanced intracellular endothelial reactive oxygen species (ROS) formation, as measured by oxidation of H2-DCF and DHE fluorescence.ConclusionsSHP-1 decreases half-life of HIF-1α under hypoxic conditions resulting in decreased cell growth due to diminished VEGF synthesis and secretion. The regulatory effect of SHP-1 on HIF-1α stability may be mediated by inhibition of endothelial ROS formation stabilizing HIF-1α protein. These findings highlight the importance of SHP-1 in hypoxic signaling and its potential as therapeutic target in ischemic diseases.

A THEMIS:SHP1 complex promotes T-cell survival.

THEMIS is critical for conventional T-cell development, but its precise molecular function remains elusive. Here, we show that THEMIS constitutively associates with the phosphatases SHP1 and SHP2. This complex requires the adapter GRB2, which bridges SHP to THEMIS in a Tyr-phosphorylation-independent fashion. Rather, SHP1 and THEMIS engage with the N-SH3 and C-SH3 domains of GRB2, respectively, a configuration that allows GRB2-SH2 to recruit the complex onto LAT. Consistent with THEMIS-mediated recruitment of SHP to the TCR signalosome, THEMIS knock-down increased TCR-induced CD3-ζ phosphorylation, Erk activation and CD69 expression, but not LCK phosphorylation. This generalized TCR signalling increase led to augmented apoptosis, a phenotype mirrored by SHP1 knock-down. Remarkably, a KI mutation of LCK Ser59, previously suggested to be key in ERK-mediated resistance towards SHP1 negative feedback, did not affect TCR signalling nor ligand discrimination in vivo. Thus, the THEMIS:SHP complex dampens early TCR signalling by a previously unknown molecular mechanism that favours T-cell survival. We discuss possible implications of this mechanism in modulating TCR output signals towards conventional T-cell development and differentiation.

SHP-1 phosphatase is a critical regulator in preventing natural killer cell self-killing.

Balance of signals generated from the engaged activating and inhibitory surface receptors regulates mature NK cell activities. The inhibitory receptors signal through immunoreceptor tyrosine based inhibitory motifs (ITIM), and recruit phosphatases such as SHP-1 to inhibit NK cell activation. To directly examine the importance of SHP-1 in regulating activities and cell fate of mature NK cells, we used our established lentiviral-based engineering protocol to knock down the SHP-1 protein expression in primary C57BL/6NCrl cells. Gene silencing of the SHP-1 in primary NK cells abrogated the ability of ITIM-containing NK inhibitory receptors to suppress the activation signals induced by NK1.1 activating receptors. We followed the fates of stably transduced SHP-1 silenced primary NK cells over a longer period of time in IL-2 containing cultures. We observed an impaired IL-2 induced proliferation in the SHP-1 knockdown NK cells. More interestingly, these “de-regulated” SHP-1 knockdown NK cells mediated specific self-killing in a real-time live cell microscopic imaging system we developed to study NK cell cytotoxicity in vitro. Selective target recognition of the SHP-1 knockdown NK cells revealed also possible involvement of the SHP-1 phosphatase in regulating other NK functions in mature NK cells.

SHP-1 As a Critical Regulator of Mycoplasma pneumoniae-Induced Inflammation in Human Asthmatic Airway Epithelial Cells

Asthma is a chronic inflammatory disease in which airway epithelial cells are the first line of defense against exposure of the airway to infectious agents. Src homology protein (SHP)-1, a protein tyrosine phosphatase, is a negative regulator of signaling pathways that are critical to the development of asthma and host defense. We hypothesize that SHP-1 function is defective in asthma, contributing to the increased inflammatory response induced by Mycoplasma pneumoniae, a pathogen known to exacerbate asthma. M. pneumoniae significantly activated SHP-1 in airway epithelial cells collected from nonasthmatic subjects by bronchoscopy with airway brushing but not in cells from asthmatic subjects. In asthmatic airway epithelial cells, M. pneumoniae induced significant PI3K/Akt phosphorylation, NF-κB activation, and IL-8 production compared with nonasthmatic cells, which were reversed by SHP-1 overexpression. Conversely, SHP-1 knockdown significantly increased IL-8 production and PI3K/Akt and NF-κB activation in the setting of M. pneumoniae infection in nonasthmatic cells, but it did not exacerbate these three parameters already activated in asthmatic cells. Thus, SHP-1 plays a critical role in abrogating M. pneumoniae-induced IL-8 production in nonasthmatic airway epithelial cells through inhibition of PI3K/Akt and NF-κB activity, but it is defective in asthma, resulting in an enhanced inflammatory response to infection.

Lyn-mediated SHP-1 recruitment to CD5 contributes to resistance to apoptosis of B-cell chronic lymphocytic leukemia cells

In B-cell chronic lymphocytic leukemia (B-CLL) cells, Lyn, a tyrosine kinase belonging to the Src family, is overexpressed and atypically localized in an aberrant cytosolic complex in an active conformation, contributing to the unbalance between cell survival and pro-apoptotic signals. In this study, we demonstrate that Lyn constitutively phosphorylates the immunoreceptor tyrosine inhibitory motifs of the inhibitory cell surface co-receptor CD5, a marker of B-CLL. As a result, CD5 provides an anchoring site to Src homology 2 domain-containing phosphatase 1 (SHP-1), a known negative regulator of hematopoietic cell function, thereby triggering the negative B-cell receptor (BCR) signaling. The subsequent segregation of SHP-1 into two pools, one bound to the inhibitory co-receptor CD5 in an active form, the other in the cytosol in an inhibited conformation, proves crucial for withstanding apoptosis, as shown by the use of phosphotyrosine phosphatase-I-I, a direct inhibitor of SHP-1, or SHP-1 knockdown. These results confirm that Lyn exhibits the unique ability to negatively regulate BCR signaling, in addition to positively regulating effectors downstream of the BCR, and identify SHP-1 as a novel player in the deranged signaling network and as a potential attractive target for new therapeutic strategies in B-CLL.

The tyrosine phosphatase, SHP-1, is involved in bronchial mucin production during oxidative stress

Mucus hypersecretion is a clinically important manifestation of chronic inflammatory airway diseases, such as asthma and Chronic obstructive pulmonary disease (COPD). Mucin production in airway epithelia is increased under conditions of oxidative stress. Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 suppression is related to the development of airway inflammation and increased ROS levels. In this study, we investigated the role of SHP-1 in mucin secretion triggered by oxidative stress. Human lung mucoepidermoid H292 carcinoma cells were transfected with specific siRNA to eliminate SHP-1 gene expression. Cultured cells were treated with hydrogen peroxide (H2O2), and Mucin 5AC(MUC5AC) gene expression and mucin production were determined. Activation of p38 mitogen activated protein kinase (MAPK) in association with MUC5AC production was evaluated. N-acetylcysteine (NAC) was employed to determine whether antioxidants could block MUC5AC production. To establish the precise role of p38, mucin expression was observed after pre-treatment of SHP-1-depleted H292 cells with the p38 chemical blocker. We investigated the in vivo effects of oxidative stress on airway mucus production in SHP-1-deficient heterozygous (mev/+) mice. MUC5AC expression was enhanced in SHP-1 knockdown H292 cells exposed to H2O2, compared to that in control cells. The ratio between phosphorylated and total p38 was significantly increased in SHP-1-deficient cells under oxidative stress. Pre-treatment with NAC suppressed both MUC5AC production and p38 activation. Blockage of p38 MAPK led to suppression of MUC5AC mRNA expression. Notably, mucin production was enhanced in the airway epithelia of mev/+ mice exposed to oxidative stress. Our results clearly indicate that SHP-1 plays an important role in airway mucin production through regulating oxidative stress.

Knockdown of protein tyrosine phosphatase SHP-1 inhibits G1/S progression in prostate cancer cells through the regulation of components of the cell-cycle machinery

SHP-1, a haematopoietic cell-specific tyrosine phosphatase, is also expressed in human prostate. In this study, we report that SHP-1 depletion in PC-3 cells induced by small interfering RNAs causes G1 phase cell-cycle arrest accompanied by changes in some components of the cell-cycle machinery. SHP-1 knockdown increases p27(Kip1) (p27) protein stability, its nuclear localization and p27 gene transcription. These effects could be mediated by PI3K-AKT pathway as SHP-1 interacts with PI3K regulating its activity and p110 catalytic subunit phosphorylation. The increase in p27 protein stability could also because of reduced cyclin-dependent kinase (CDK2) activity. SHP-1 knockdown decreases the CDK6 levels, inducing retinoblastoma protein hypophosphorylation, downregulation of cyclin E and thereby a decrease in the CDK2 activity. However, the codepletion of SHP-1 and p27 does not produce re-entry into the cycle, implying that p27 is not required to maintain cell-cycle arrest induced by SHP-1 depletion. The maintenance of the PC-3 cell anti-proliferative response after p27 loss could be because of mislocalization of CDK2 induced by SHP-1 knockdown. This study shows that SHP-1 depletion promotes cell-cycle arrest by modulating the activity of cell-cycle regulators and suggests that SHP-1 may be required for the proper functioning of events governing cell-cycle progression.

Simulation and prediction of endothelial cell adhesion modulated by molecular engineering

Computational modeling is an effective approach for simulating and predicting the development and remodeling of biological systems. Here, we propose to integrate a probabilistic mathematical modeling approach into an emerging bioengineering area known as molecular regenerative engineering. This experimental–computational framework can be used to simulate and predict the efficacy of molecular engineering in controlling and enhancing the regenerative processes of pathologically disordered cells, tissues and organs. We demonstrate the significance and principles of this approach by using a special case as an example: enhancement of endothelial cell adhesion to arterial constructs by siRNA-mediated modulation of an adhesion-inhibiting factor known as Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1. A continuum approach is used to model endothelial cell adhesion at the cellular level, while the Kramers reaction-rate theory is used to model cell adhesion at the subcellular and molecular levels. We show that the proposed computational model can potentially be used to simulate the processes and alterations of endothelial cell adhesion at the molecular and cellular levels under shear flow and to predict the effectiveness of siRNA-mediated SHP-1 knockdown in enhancing endothelial cell adhesion in arterial constructs. These preliminary investigations suggest that this integrated experimental–computational approach may be used for the simulation and prediction of regenerative processes in response to molecular engineering.

Formation of smooth muscle α actin filaments in CD34+ bone marrow cells on arterial elastic laminae: Potential role of SH2 domain-containing protein tyrosine phosphatase-1

Arterial smooth muscle cells (SMCs) are present in the elastic lamina-containing media, suggesting that the elastic laminae may regulate the development of SMCs. Here, we investigated the role of elastic laminae in regulating the formation of SM α actin filaments in mouse CD34+ bone marrow cells and the role of a protein tyrosine phosphatase, SH2 domain-containing protein tyrosine phosphatase (SHP)-1, in the mediation of this process. Mouse CD34+ bone marrow cells were isolated by magnetic separation and used for assessing the influence of elastic laminae and collagen matrix on the formation of SM α actin filaments. CD34+ cells with transgenic SHP-1 knockout or siRNA-mediated SHP-1 knockdown were used to assess the role of SHP-1 in mediating the formation of SM α actin filaments. In cell culture tests, elastic laminae, but not collagen matrix, stimulated the formation of SM α actin filaments in CD34+ cells. The phosphatase SHP-1 mediated the stimulatory effect of elastic laminae. The interaction of CD34+ cells with elastic laminae, but not with collagen matrix, induced activation of SHP-1. The suppression of SHP-1 by transgenic SHP-1 knockout or siRNA-mediated SHP-1 knockdown significantly reduced the formation of SM α actin filaments in CD34+ cells cultured on elastic laminae. The in vitro observations were confirmed by using an in vivo model of implantation of elastic lamina and collagen matrix scaffolds into the aorta. These observations suggest that elastic laminae stimulate the formation of SM α actin filaments in CD34+ bone marrow cells and SHP-1 mediates the stimulatory effect of elastic laminae.